Method for Making Targeted Therapeutic Agents

ABSTRACT

Provided herein are methods and kits for making a targeted therapeutic for treating a disease or condition. The therapeutic agents can be targeted to patient-specific disease markers. In one of these methods, the method includes obtaining a biological sample from a patient having the disease or condition, or who is at risk for developing the disease or condition. In this particular method, the sample includes a population of diseased cells, screening a library comprising proteins linked to their cognate mRNAs to identify mRNA-protein pairs that bind to the diseased cells, isolating one or more proteins from the identified mRNA-protein pairs, and conjugating the isolated protein(s) to a therapeutic agent. Some of the methods further include preparing a library with proteins linked to their cognate mRNAs. In certain of these methods, the preparation of the library includes providing at least two candidate mRNA molecules in which each of the mRNA molecules includes a cross-linker, translating at least two of the candidate mRNA molecules to generate at least one translated protein, and linking at least one of the candidate mRNA molecules to its corresponding translated protein via the cross-linker to form at least one cognate pair.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for making therapeuticstailored to individual patients or sub-populations of patients, as wellas methods of using such therapeutics to treat malignancies, pathogenicinfections, and other conditions, and to reduce or prevent transplantrejection.

2. Description of the Related Art

Many malignant cells display epitopes that are specific not only to thetype of malignancy but also to the individual patient. In some aspects,the present invention is directed to therapeutics that can be targetedto patient-specific epitopes, such as those displayed on malignantlymphocytes.

Lymphocytes are critical to the immune system of vertebrates.Lymphocytes are produced in the thymus, spleen and bone marrow (adult)and represent about 30% of the total white blood cells present in thecirculatory system of humans (adult). There are two majorsub-populations of lymphocytes: T cells and B cells. T cells areresponsible for cell-mediated immunity, while B cells are responsiblefor antibody production (humoral immunity). In a typical immuneresponse, T cells are activated when the T cell receptor binds tofragments of an antigen that are bound to major histocompatibilitycomplex (“MHC”) glycoproteins on the surface of an antigen presentingcell; such activation causes release of biological mediators(“interleukins”) which, in essence, stimulate B cells to differentiateand produce antibody (“immunoglobulins”) against the antigen.

The etiology of hematological cancers such as lymphomas, leukemias andmultiple myelomas varies or is unknown. Suspected causes range fromviral and chemical exposure to familial propensities. A commondenominator in these cancers however, is that they all begin with amalignantly transformed B-cell or T-cell which divides to form a cloneof cells that express the same Fab idiotype on the immunoglobulinproteins they express on their surface. One of the difficulties intreating these cancers is that each cancer expresses a unique idiotype.Developing a therapeutic treatment that effectively and selectivelytreats all possible idiotypes has therefore been elusive.

Conventional treatments for hematological cancers typically involveprocedures that destroy all blood producing cells in the bone marrow,including the malignant clone, followed by bone marrow replacement withstem cells isolated from the patient or bone marrow from a matched donorto reconstruct the blood producing system. These treatments are highlyinvasive and marginally curative. One approach involves treatment withmonoclonal antibody vaccines that recognize cell surface proteinsutilized as “markers” for identification. Therapeutics adopting thisapproach include Compath-H (Alemtuzumab), HLL2 (Epartuzumab), Hu1D10,and Rituximab, (e.g., U.S. Pat. No. 6,455,043). However, a seriouslimitation with these monoclonal antibody based therapeutics is that thetargeted cell surface antigens are often found on both normal as well asmalignant cells. In addition, because of the difficulties in producinghuman monoclonal antibodies, monoclonal antibody vaccines typicallyutilize “Chimeric” antibodies, i.e., antibodies which comprise portionsfrom two or more different species (e.g., mouse and human). Repeatedinjections of such foreign antibodies can lead to the induction ofimmune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody response (“HAMA” response). Patients may alsodevelop a Human Anti-Chimeric Antibody response (“HACA” response). HAMAand HACA can attack “foreign” antibodies so that they are, in effect,neutralized before they reach their target site(s). A further drawbackto monoclonal antibody vaccines is the time and expense required toproduce monoclonal antibodies. This is particularly problematicconsidering that targeted epitopes, such as CD20, CD19, CD52w, andanti-class II HLA can readily mutate to form new tumors that areresistant to previous therapeutics (see e.g., Clinical Cancer Research,5:611-615, 1999). Thus, there is a need in the art for effective, lowcost therapeutics for treating malignancies by selectively targeting anindividual's cancerous cells over benign cells.

Like malignant cells, the cells of transplanted tissues and organsdisplay cell-surface epitopes that are differentially expressed intransplanted cells relative to native cells. In some aspects, thepresent invention is directed to therapeutics that can be targeted tothe cells of transplanted tissues or organs by recognition of suchvariable epitopes. Transplant rejection is caused by an immune responseto alloantigens on the transplanted cells, which are proteins specificfor an individual patient (including the donor), and which are thusperceived as foreign by the recipient. The most common alloantigensinvolved in transplant rejections are MHC (major histocompatibilitycomplex) molecules, which are expressed on the surface of transplantedcells and are highly polymorphic among individuals. Foreign MHCmolecules are recognized by the recipient's immune system, causing animmune response that leads to rejection of the transplant.

One pathway through which the immune system rejects transplanted tissuesis complement-mediated immunity, which can be activated by binding of C1(a first component of complement) to an immune complex consisting of anMHC antigen on a transplanted cell and the recipient's natural antibodyagainst the MHC antigen. Activation of the pathway results in theassembly of enzymes called C3 convertases, which cleave the complementcomponent C3 to form C3a and C3b. Some of the C3b molecules then bind tothe C3 convertases to cleave C5 to C5a and C5b. The biologicalactivities of the complement system, in turn, are derived from thecleavage products of C3 and C5. Another subcomponent of the complementsystem, C1q, is involved in the initial steps of complement activation.To date, methods for treating transplant rejection by modulatingcomplement-mediated immunity have suffered from side effects associatedwith non-selectivity, due, for example, to the suppression of allcomplement-mediated immune responses by a therapeutic agent, whicheliminates an important component of the immune system's ability toprotect against foreign molecules.

Accordingly, there is a need in the art for effective, low costtherapeutics for reducing or preventing transplant rejection byselectively inhibiting the body's immune response against transplantedcells while retaining protection against foreign pathogens, and/or byselectively destroying particular cell types that stimulate a largerimmune response.

SUMMARY OF THE INVENTION

In various aspects, methods are provided herein for developing targetedtherapeutics useful in treating a wide range of conditions by targetingcell-surface markers (e.g., epitopes, idiotypes, and the like) or othermolecules that are differentially expressed by, or in close proximityto, malignant cells, pathogens, transplanted cells, and/or otherentities targeted for treatment. Also provided are methods for treatinga disease or condition by administering a therapeutic produced bymethods described herein.

In some preferred embodiments, methods provided herein utilize noveltechniques for linking proteins to their corresponding mRNAs, andscreening the protein-mRNA complexes for binding to a molecular targetassociated with one or more etiological determinants. In variouspreferred embodiments, therapeutics provided herein are designed torecognize molecular targets that are differentially expressed in anindividual patient seeking treatment, or in a sub-population ofpatients, such as patients diagnosed with a specific strain or subtypeof a disease or condition. Proteins having high affinity for a target ofinterest are preferably isolated and linked to one or more therapeuticagents effective against the disease being treated (e.g., cytotoxicagents), to produce a variety of targeted therapeutics. Advantageously,the rapid and efficient identification, isolation, and production ofproteins capable of recognizing targets of interest provides effective,low cost methods for the production of patient- and/or disease-specifictherapeutics. In various embodiments, methods provided hereinbeneficially allow a wide range of diseases and conditions to be treatedwith tailored therapeutics, within the context of existing health carebudgets and resource allocations.

In various aspects, methods are provided herein for producing tailoredtherapeutics for treating cancers and other conditions, wherein thetailored therapeutics comprise a “targeting domain” that binds to amolecular target associated with a disease or condition selected fortreatment, and a “therapeutic agent” capable of treating or preventingsaid disease or condition. In some preferred embodiments, the targetingdomain is tailored to recognize targets that are differentiallyexpressed in particular patients or sub-populations of patients, whilein these and/or other embodiments, the therapeutic agent does notrequire substantial tailoring to individual patients or sub-populationsof patients. This “modular” architecture advantageously allows for thecreation of individualized therapeutics by tailoring only the smallportion of the administrable therapeutic comprising the targetingdomain, which can then be used to enhance the efficacy of a variety ofpre-existing or easily prepared therapeutic agents.

In some preferred embodiments, the targeting domain is tailored to bindan epitope selectively or preferentially expressed by cancerous cellsrelative to non-cancerous cells in a patient seeking treatment, and thetherapeutic agent is an antibody or other molecule (hereinafter referredto as the “immune effector”) capable of stimulating an immune responsein the patient. In some preferred embodiments, the targeted epitopes aresubstantially absent from non-cancerous cells, and the therapeutic agentdoes not otherwise substantially bind to non-cancerous cells. In somepreferred embodiments, the cancer is a hematological cancer, such as alymphoma (e.g., non-Hodgkin's lymphoma), a leukemia, or a multiplemyeloma, wherein cancerous and/or malignant cells express apatient-specific epitope that can be an idiotype.

In various embodiments, the therapeutic agent is linked, fused, orderivatized, directly or indirectly, to the targeting domain to form themodular therapeutic. In some embodiments, an immune effector iscovalently linked to a target-binding domain, while in other embodimentsit is non-covalently bound. In some embodiments, the target-bindingdomain is part of a bifunctional protein comprising a target-bindingdomain fused to a second domain that binds the immune effector. Theimmune effector-binding domain may comprise an epitope recognized byvariable regions of an antibody, or a molecule that binds other regionsof an antibody, such as the Fc region. The immune effector-bindingdomain may also comprise a peptide sequence designed to tightly bind theimmune effector (see e.g., FIG. 9). The bifunctional protein maycomprise a fusion protein, or the two domains can be covalently ornon-covalently linked. The target-binding domain and the immune effectorbinding domain can be directly linked, or indirectly linked, for examplevia a flexible linker peptide.

In other aspects, the invention provides methods for preparing atherapeutic for treating a cancer comprising isolating complexes ofexpressed mRNA molecules and their nascent polypeptides from an mRNAexpression library; screening the protein-mRNA complexes for binding toa molecular target associated with an etiological determinant, such asan epitope displayed by a cancerous cell; isolating and expressing mRNAencoding a protein that binds the target epitope; and derivatizing thetarget epitope-binding protein (or derivatives, fragments or subunitsthereof) to a therapeutic agent, such as an antibody capable ofeliciting an immune response against the target. In some embodiments,the preparation of the therapeutic further comprises allowing isolatedmRNA encoding a target-binding domain to undergo in vitro evolution,selective mutagenesis, and/or other methods known in the art to identifyand isolate mRNAs exhibiting stronger or more effective binding to thetarget epitope, as described in more detail below.

In yet further aspects, the invention provides methods for treating adisease or condition, such as a cancer, comprising identifying a targetepitope or other molecule specifically or preferentially expressed on,or in close proximity to, an etiological determinant of the conditiontargeted for treatment, in a patient in need of treatment; isolating aprotein that binds the target, but does not bind substantially tonon-targets; linking the target-binding protein (or derivatives,fragments or subunits thereof) to a therapeutic agent, such as anantibody capable of eliciting an immune response in the subject ofinterest; and administering the therapeutic to the patient in an amountand for a time sufficient to treat the condition targeted for treatment.

In a still further aspect, the invention provides methods foridentifying proteins that bind cancer cell target epitopes, or otheretiological determinants, comprising isolating complexes of expressedmRNA molecules and their nascent polypeptides from an mRNA expressionlibrary; screening the protein-mRNA complexes for binding to a targetepitope displayed by a cancerous cell; isolating and expressing mRNAencoding a protein that binds the target epitope; and derivatizing thetarget epitope-binding protein (or derivatives, fragments or subunitsthereof) to an antibody capable of eliciting an immune response. In someembodiments, the methods may further comprise allowing isolated mRNAencoding a target epitope-binding protein to undergo in vitro evolution,selective mutagenesis, or other methods known in the art to identify andisolate mRNAs exhibiting stronger or more effective binding to thetarget epitope.

In yet another aspect, the invention provides a kit for developing anindividualized therapeutic for the treatment of a disease or conditioncharacterized by the expression of disease- and/or patient-specificetiological determinants. In some preferred embodiments, a kit isprovided for developing patient-specific therapeutics for the treatmentof a cancer, including solid tumors and hematological cancers, whereinthe therapeutics are targeted to a unique cell-surface epitopedifferentially expressed on the surface of cancerous and/or malignantcells.

In an additional aspect, the invention provides therapeutics and methodsfor reducing or preventing transplant rejection. In some embodiments,the target-binding protein binds to cell surface antigens displayed bytransplanted cells, such as MHC antigens, and the therapeutic agent(e.g., the immune effector) comprises a protein or other molecule thatbinds to and inhibits one or more molecular determinants of the immuneresponse. In some preferred embodiments, the immune effector binds theC1q or C3 components of the complement system to thereby inhibit theactivation of complement-mediated immunity. In other embodiments, theimmune effector stimulates an immune response to eliminate transplantedcells bearing “foreign” MHC or other antigens. In further embodiments,the therapeutic agent ablates or prevents the clonal expansion oflymphocyte subpopulations expressing specific epitopes, for exampleepitopes that recognize MHC antigens.

The invention further provides methods and kits for inhibitingtransplant rejection that are essentially similar to those describedabove for treating malignancies.

Further aspects of the invention provide therapeutics, methods and kits,essentially similar to those described above, wherein the target-bindingdomain is selected to bind one or more variable epitopes expressed by adisease-causing pathogen, or one or more cells infected by adisease-causing pathogen, and the therapeutic agent comprises ananti-pathogenic drug, such as an antibiotic or other cytotoxic agent. Insome embodiments, the pathogen is a virus, such as HIV.

In various aspects, methods provided herein allow for the rapid andcost-effective creation of individualized therapeutics. The followingdetailed description illustrates various aspects of the invention asthey relate to particular applications. However, the description appliesequally to the development and use of therapeutics and methods for thetreatment of a wide variety of conditions, including but not limited to,the treatment of malignancies, the reduction and elimination ofpathogens, the reduction and/or prevention of transplant rejection, andin general, the treatment of any condition involving a therapeutictarget which exhibits differential binding characteristics relative tonon-targeted cells or molecules.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Complex comprising a target epitope-binding protein and animmune effector antibody reacted with a malignant cell via the Fabidiotype expressed on the cell surface.

FIG. 2. Illustration of a known human antibody (Ab) to a small humanprotein (Ebbd). The protein can serve as an epitope that links theantibody (Ab) to the therapeutic. The role of the Ab is to act as animmune effector that ultimately triggers an immune response.

FIG. 3. Illustration of scaled up production of mRNA encoding an immuneeffector-binding protein (Ebbd) for ligation purposes in construction ofoligonucleotide sequence that codes for the bifunctional proteincomponent of the therapeutic.

FIG. 4. ProteoNova's system for breeding a protein that bindsselectively to an epitopes expressed on the surface of certain cancercells. The system includes in vitro translation of an mRNA library togive protein libraries in which each protein remains linked with itscognate mRNA. Selection includes steps to identity proteins thatselectively bind the cancer epitopes but not the epitopes expressed bynormal cells. The selection methods used are dependent on the epitopesisolation and display procedure used. The system also includes rapiddirected evolution, selection, and production, in quantity, of theprotein(s) with targeted properties.

FIG. 5. Process for isolating the mRNA from the protein that binds theidiotype or target epitope on malignant cells. The illustration excludesnegative selection.

FIG. 6. Bifunctional protein translated from an oligonucleotide sequencecreated by the ligation of the mRNAs that code for a targetepitope-binding protein and for a human protein (the immune effectorbinding domain (Ebbd)) that is bound by a human antibody.

FIG. 7. Individualized cancer therapeutic prepared by forming a complexthat includes a bifunctional protein with epitopes for the immuneeffector Ab (Anti-Ebbd antibody) and the Fab idiotype or target epitope.

FIG. 8. Illustration of a malignant cell tagged by the therapeuticthrough the linkage between the idiotype or target epitope and theprotein that binds the iidiotype or target epitope. The exposed humanantibody triggers the curative immune response.

FIG. 9 Therapeutic comprising a first peptide (the targetepitope-binding domain) bred to bind to the target epitope and a secondpeptide (the immune effector-binding domain) bred to bind a stable C3convertase (the immune effector). Binding of the therapeutic to thetarget epitope elicits a complement-mediated immune response at thebinding site.

FIG. 10 Therapeutic comprising a first peptide (the targetepitope-binding domain) bred to bind to the target epitope and a secondpeptide (the immune effector) bred to bind the C1 component of thecomplement system. Binding of the therapeutic to the target epitopeelicits binding of endogenous C1 to the effector, and the initiation ofa complement-mediated immune response at the binding site.

FIG. 11 illustrates schematically one example of the complex formed bythe mRNA and its protein product when linked by a modified tRNA oranalog. As shown, a codon of the mRNA pairs with the anticodon of amodified tRNA and is covalently crosslinked to a psoralen monoadduct, ora non-psoralen crosslinker or aryl azides by UV irradiation. Thetranslated polypeptide is linked to the modified tRNA via the ribosomalpeptidyl transferase. Both linkages occur while the mRNA and nascentprotein are held in place by the ribosome.

FIG. 12 illustrates schematically an example of the in vitro selectionand evolution process, wherein the starting nucleic acids and theirprotein products are linked (e.g., according to FIG. 1) and are selectedby a particular characteristic exhibited by the protein. Proteins notexhibiting the particular characteristic are discarded and those havingthe characteristic are amplified with variation, preferably viaamplification with variation of the mRNA, to form a new population. Invarious embodiments, nonbinding proteins will be selected. The newpopulation is translated and linked via a modified tRNA or analog, andthe selection process is repeated. As many selection andamplification/mutation rounds as desired can be performed to optimizethe protein product.

FIG. 13 illustrates one method of construction of a tRNA molecule of theinvention. In this embodiment, the 5′ end of a tRNA, a nucleic acidencoding an anticodon loop and having a molecule capable of stablylinking to mRNA (such as psoralen, as used in this example), and the 3′end of tRNA modified with a terminal puromycin molecule are ligated toform a complete modified tRNA for use in the in vitro evolution methodsof the invention. Other embodiments do not include puromycin.

FIG. 14 describes two alternative embodiments by which the crosslinkingmolecule psoralen can be positioned such that it is capable of linkingthe mRNA with the tRNA in the methods of the invention. A firstembodiment includes linking the crosslinker (e.g., psoralen monoadduct)to the mRNA, and a second embodiment includes linking the crosslinker tothe anticodon of the tRNA molecule. The crosslinker can either bemonoadducted to the anticodon or the 3′ terminal codon of the readingframe for known or partially known messages. This can be done in aseparate procedure from translation, e.g., before translation occurs.

FIG. 15 illustrates the chemical structures for uridine andpseudouridine. Pseudouridine is a naturally occurring base found in tRNAthat forms hydrogen bonds just as uridine does, but lacks the 5-6 doublebond that is the target for psoralen.

FIG. 16 illustrates some embodiments of the present invention. The SATA,Linking tRNA Analog and Nonsense Suppressor analog, in certainembodiments, are shown.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The terms “T lymphocyte” and “T cell” as used herein encompass any cellwithin the T lymphocyte lineage from T cell precursors to mature Tcells.

The terms “B lymphocyte” and “B cell” encompasses any cell within the Bcell lineage from B cell precursors, such as pre-B cells, to mature Bcells and plasma cells.

Immunoglobulin molecules consist of heavy (H) and light (L) chains,which comprise highly specific variable regions at their amino termini.The variable (V) regions of the H (V_(H)) and L (V_(L)) chains combineto form the unique antigen recognition or antigen combining site of theimmunoglobulin (Ig) protein. The variable regions of an Ig moleculecontain determinants (i.e., molecular shapes) that can be recognized asantigens or idiotypes.

The term “epitope” refers to the set of antigenic or epitopicdeterminants (i.e., idiotopes) of an immunoglobulin V domain (i.e., theantigen combining site formed by the association of the complementaritydetermining regions or V_(H) and V_(L) regions).

The term “idiotope” refers to a single idiotypic epitope located along aportion of the V region of an immunoglobulin molecule.

The term “immune effector” refers to a molecule, or derivatives,fragments, or subunits thereof, able to stimulate an immune response inthe subject being treated, and may comprise an antibody, or derivatives,fragments, or subunits thereof, or a non-antibody molecule.

An “adjuvant” is a compound which enhances or stimulates the immuneresponse when administered with one or more antigen(s).

“Malignant cells” refers to cells, which if left untreated, give rise toa cancer.

The terms “protein,” “peptide,” and “polypeptide” are defined herein tomean a polymeric molecule of two or more units comprised of amino acidsin any form (e.g., D- or L-amino acids, synthetic or modified aminoacids capable of polymerizing via peptide bonds, etc.), and these termsmay be used interchangeably herein.

Provided herein, are methods for producing individualized therapeuticsthat are tailored to specific patients, or to sub-populations ofpatients suffering from a particular disease or condition. In variousembodiments, the therapeutics are comprised of a “modular” architecturethat allows tailoring of a small protein domain (the “target-bindingdomain”) to bind one or more patient- and/or disease-specific markers,and use of the tailored domain to direct a variety of existing or easilyproduced therapeutics that are effective against the condition targetedfor treatment. The targeted disease markers can comprise any type ofmolecule, or portion or derivative thereof, or complex of molecules,including but not limited to, proteins, nucleic acids, lipids, chemicalcompounds, polymers, and metals, as well as biological structures, suchas cell membranes, cytoskeletal elements, receptors, and even entirecells. Advantageously, the markers are expressed on, or in closeproximity to, an etiological determinant of the condition targeted fortreatment, and activity of the therapeutic agent is focused todisease-causing cells, pathogens, proteins, and/or other determinants ofthe condition being treated.

In some embodiments, the modular therapeutic is tailored for thetreatment of a hematological cancer, and designed to bind to the uniqueFab idiotype on the surface of malignant B-cells and/or T-cells. Forexample, in some preferred embodiments, a therapeutic is provided forthe treatment of non-Hodgkin's Lymphoma (NHL), which is a “clonal”B-cell disease, in which all cancerous cells are derived from a single,malignant B-cell. As a result, every NHL cell expresses a commonidiotype (comprising the variable domains of surface expressed IgMmolecules) that is unique to each patient, which can be targeted by thetarget-binding portions of the modular therapeutics provided herein.B-cells can be isolated from the lymph nodes, or from peripheral blood,using methods known in the art. For example, in some embodiments,erythrocytes and/or granulocytes may be separated from the B-cells bycentrifugation in a liquid having a density intermediate between thegroups of cells to be separated. Means of obtaining T-lymphocytes arealso well known in the art, such as isolation from the peripheral bloodof a patient, and separation on the basis of size and/or density.Extraction of proteins from B-cells and/or T-cells may be performed byany of the many means known in the art. For example, cells may be lysedby a detergent or by mechanical means. If desired, nucleic acids can beremoved from the cell preparation by enzymatic digestion or byprecipitation with agents such as streptomycin. Such means are wellknown in the art.

The mechanism of action of a modular therapeutic for treating ahematological cancer prepared according to methods described herein isillustrated in FIG. 1. The administered therapeutic binds to the uniqueFab idiotype on the surface of malignant B-cells and/or T-cells, and theetiological determinant of the hematological cancer, with which theidiotype is associated, comprises malignant and/or cancerous cellsexpressing the targeted idiotype. Binding of the targeting domain to theidiotype effects an immune response that destroys the malignant cells.By itself, the target-binding protein is too small to elicit an immuneresponse, and thus the bound immune effector will not produce an IR inthe absence of binding to the targeted idiotype. When the target-bindingprotein binds to the Fab idiotype on the surface of a malignant cell,the malignant cell acts as a carrier that confers immunogenicity to thetarget epitope-binding protein, allowing the immune effector to producean IR that targets the malignant cell. In further embodiments,therapeutics and methods of the invention can also be used to targetepitopes that distinguish non hematological cancers.

In various preferred embodiments, targeted markers are differentiallyexpressed (i) in an individual patient, or a defined sub-group ofpatients, relative to other patients having similar diagnoses, and/or(ii) in association with cells or other molecular targets associatedwith the etiology of the condition, relative to cells/molecules that areunassociated with the condition and preferably not subjected to thetherapeutic agent. Advantageously, the selectivity of tailoredtherapeutics enhances the efficacy of treatment relative to existing,non-tailored therapeutics, due, for example, to the non-selectiveactivity of non-tailored therapeutics against healthy cells, and/orfailure of existing targeted therapeutics to account for inter-patientvariability in the targeted marker(s). For example, in the case ofhematological cancers, the targeted idiotype is unique to both thepatient and to the malignant cells, allowing the activity of thetherapeutic agent (effecting an immune response) to be selectivelydirected to targeted cells, sparing non-cancerous cells. Moreover,because the idiotype is unique to each patient, non-tailoredtherapeutics would result in a non-selective, or less-selective,therapeutic response.

Examples of cell-surface markers differentially expressed by malignantcells include, but are not limited to: for fluid tumors, stable cellsurface antigen epitopes, such as CD-20 and CD-22, and for solid tumors,surface epitopes, such as CD-19 and CD-33, which become internalizedupon binding with a mAb. Other differentially expressed cell-surfacemarkers are known in the art, including but not limited to, CD-52w andclass II HLA antigens. In some preferred embodiments, the targetedepitope is a cancer cell-specific epitope that is mutated (see e.g.,Clinical Cancer Research, 5:611-615, 1999) in the patient targeted fortreatment. Advantageously, methods provided herein allow for moreefficacious treatment of cancers by allowing the development ofindividualized therapeutics that target mutated epitopes, which areunique to each patient.

In some preferred embodiments (as shown, e.g., in FIG. 2), thetarget-binding domain of the modular therapeutic comprises a firstsub-domain that binds the target (e.g., a target epitope-binding domain(Tebd)), and a second sub-domain that binds the therapeutic agent (e.g.,an immune effector-binding domain (Ebbd)). In some embodiments, thetherapeutic agent is an agent that is capable of stimulating an immuneresponse in the subject targeted for treatment, such as an antibody, ora derivative, fragment, or subunit thereof, and the domain that bindsthe therapeutic agent is a small protein recognized by the therapeuticagent, for example an epitope recognized by a therapeutic agentantibody. Well-characterized antibody-antigen pairs can also be utilizedthat are known in the art, and are commercially available. Antibodiesuseful in the invention may be derived from any mammal, or may be achimeric antibody derived from a combination of different mammals. Themammal may be, for example, a rabbit, a mouse, a rat, a goat, or ahuman. The antibody is preferably a human antibody. Reactivity ofantibodies against a target antigen may be established by a number ofwell known means, including Western blot, immunoprecipitation, ELISA,and FACS analyses using, as appropriate, Fab idiotype fragments,peptides, idiotype-expressing cells or extracts thereof. The antibodycan belong to any antibody class and/or sub-class. The antibodies mayalso contain fragments from antibodies of different classes andsub-classes, thereby forming a composite.

In various embodiments, human monoclonal antibodies having a desiredbinding activity are produced using methods known in the art (forreview, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539), forexample by screening a phage display library, as described, e.g., inParmley and Smith Gene 73:305-318 (1988), Barbas et al., Proc. Natl.Acad. Sci. USA 88: 7978-7982 (1991), Griffiths et al., EMBO J. 13:3245-3260 (1994), Griffiths and Hoogenboom, Building an in vitro immunesystem: human antibodies from phage display libraries. In: ProteinEngineering of Antibody Molecules for Prophylactic and TherapeuticApplications in Man. Clark, M. (Ed.), Nottingham Academic, pp 45-64(1993), and Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82, all of which are herein incorporated byreference. Typically, clones corresponding to antibodies which producebinding affinities of a desired magnitude are identified, and the DNA isused to produce the antibodies of interest using standard recombinantexpression methods.

Fully human monoclonal antibodies may also be produced using transgenicmice engineered to contain human immunoglobulin gene loci, as describedin PCT Patent Application WO98/24893 and Jakobovits, 1998, Exp. Opin.Invest. Drugs 7 (4): 607-614, herein incorporated by reference. Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

In some embodiments, an antibody against an antigen of interest, such asan immune effector-binding domain, is produced in the patient selectedfor treatment with the modular therapeutic. For example, in someembodiments, the patient is “vaccinated” with the antigen of interest,and antibodies from the patient against the antigen are selected bybinding to the antigen of interest, as described in Zebedee, et al.Proc. Natl. Acad. Sci. USA 89: 3175-3179 (1992), Burton et al., Proc.Natl. Acad. Sci. USA 88: 10134-10137 (1991), and Barbas et al., Proc.Natl. Acad. Sci. USA 89: 10164-20168 (1991).

In some embodiments, additional rounds of screening are performed toincrease the affinity of the originally isolated antibody. For example,in some embodiments, the affinity of the antibody is enhanced byaffinity maturation, in which hypervariable antibody regions are mutatedto produce a large number of combinations, and the correspondingantibody variants are screened via phage display to select antibodieshaving the desired affinity for the antigen. In further embodiments, thesmall protein epitope can undergo in vitro evolution, as described inmore detail below, to increase its binding affinity for the antibody.Advantageously, in some embodiments, for example those in whichantibodies are produced by “vaccinating” a patient, an analogous processis carried out by the host immune system (e.g., via clonal selection) toproduce high affinity antibodies specific for the antigen of interest.

In other embodiments, the immune effector comprises a non-antibodymolecule capable of stimulating an immune response in the subjectreceiving treatment. For example, the immune effector may comprise a C3convertase, as described, e.g., in U.S. Pat. No. 6,268,485 to Farriesand Harrison, which is herein incorporated by reference. C3 convertaseis an enzyme that catalyzes the proteolytic conversion of C3 proteininto C3a and C3b, which conversion comprises a critical step in thegeneration of the complement system response. C3b binds to cell surfacesnear its site of generation, where it mediates phagocytosis and otherdestructive immune responses. In some embodiments, C3 convertase ismodified or derivatized to confer reduced susceptibility to inhibitors,resistance to proteolytic cleavage, enhanced affinity for cofactors, orother modifications that enhance the effectiveness of the enzyme instimulating a complement-mediated immune response. In some embodiments,the immune effector comprises a C5 convertase, a mannose binding protein(MBP), or another molecule that stimulates complement-mediated immunity.

Linking C3 convertase to the therapeutics of the instant inventionallows a complement-mediated immune response to be directed to cells,pathogens, pathogen-infected cells, and/or other cells targeted fortreatment by binding of the target epitope binding protein to variableepitopes on the surface of the targeted cells. A therapeutic with C3convertase as the immune effector is illustrated in FIG. 9. In apreferred embodiment, peptide sequences encoding proteins that bind C3convertase (the immune effector binding protein) and a target epitope(the target epitope binding protein) are identified, isolated, andoptionally bred by in vitro evolution or other techniques to increasetheir affinity to their target ligands. A bifunctional fusion protein isthen constructed comprising the target epitope binding domain, and theconvertase binding peptide as the immune effector binding domain (Ebbd).The therapeutic is administered to a patient in need of treatment, uponwhich the target epitope-binding domain binds to the surface of amalignant cell or other target, and the C3 convertase produces acomplement-mediated immune response, which includes the production ofmembrane attack complexes that mediate the destruction of the targetedcell.

In some embodiments, the therapeutic comprises a target epitope-bindingdomain and a C3 convertase binding domain. Upon being administered, suchtherapeutics bind a target epitope and recruit endogenous C3 convertaseto initiate a complement-mediated immune response. Such an approach isillustrated in FIG. 10, which shows a therapeutic in which the immuneeffector binds the C1 component of the complement system. Upon beingadministered to a patient in need of treatment, the therapeutic binds tothe target epitope and recruits endogenous C1 to the cell surface, whereit induces a complement-mediated immune response.

In other embodiments, the immune effector component of the therapeuticbinds to and inhibits C1q, C3, C5, and/or other molecules involved ingenerating complement-mediated immune responses, such as the proteinsdescribed in U.S. Pat. No. 5,650,389. Upon being administered to apatient in need of treatment, such therapeutics bind to the targetepitope and inhibit the immune response that would otherwise be directedat or near the binding site. For example, in some preferred embodiments,a patient is the recipient of transplanted cells (e.g., stem cells, orcells comprising a transplanted organ), and the targeted therapeutic iscapable of recognizing transplanted cells, and inhibiting an immuneresponse against such cells. In further embodiments, targetedtherapeutics for the inhibition of transplant rejection comprise atarget-binding domain that targets one or more epitopes of immune cellsthat recognize transplanted cells, for example epitopes that bind MHCantigens on the surfaces of transplanted cells, and the therapeuticagent is an immune effector capable of stimulating an immune response inthe patient against the anti-transplant immune cells.

Advantageously, methods provided herein are rapid and cost-effectiveenough to allow routine creation of individualized therapeutics.Therapeutics according of the invention can preferably be created, fromreception of the biopsy material to the completion of the therapeutic,in a matter of weeks and at a cost of several thousand dollars.

A bifunctional fusion protein for use in the invention can be producedaccording to standard recombinant DNA techniques known to those skilledin the art (e.g., Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989)).In some embodiments, the purified bifunctional protein can be reactedwith the immune effector, yielding a conjugated effector-bifunctionalprotein complex, as shown FIG. 7, which comprises the therapeutic. Thebifunctional protein can also be chemically bound to the immune effectorthrough means known in the art.

In some embodiments, a protein coupling agent, such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), or bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene), is used to link two or more proteincomponents comprising the therapeutic.

In some embodiments, a recombinant fusion protein is prepared by:linking a first polynucleotide sequence encoding atarget-epitope-binding protein, or derivatives, fragments or subunitsthereof, to a second polynucleotide sequence encoding an epitope orother protein that binds the immune effector to generate a chimericcoding sequence; subcloning the chimeric coding sequence into anexpression vector; transfecting a cell with the expression vector; andpurifying the fusion protein expressed by the transfected cell. In someembodiments, the chimeric polynucleotide can include an initiationsequence appropriate for prokaryotic and/or eukaryotic in vitrotranslation systems, and/or a selectable marker.

In some embodiments, the bifunctional protein complex is made by invitro translation of an mRNA oligo that includes the mRNA encoding bothproteins and, in some embodiments, the major histocompatibility complexI and/or II. The cDNA for the mRNA sequences can be synthesized orobtained commercially, and transcribed by PCR to obtain sufficient mRNA,as shown in FIG. 3. In various embodiments, the oligo is formed byligating mRNA encoding the immune effector-binding domain and the mRNAencoding the target epitope-binding domain. In some cases this fusioncan be connected through an mRNA bridge that codes for hydrophilic aminoacids, as illustrated in FIG. 6. The mRNA can then be translated invitro using prokaryotic or eukaryotic translation systems, and theresultant bifunctional protein can purified by gel electrophoresis orany other method known in the art.

The present fusion proteins can also comprise one or more numerous othercomponents that enhance the utility of the chimeric proteins. Forexample, the proteins can be designed to contain an epitope tag useful,for example, to facilitate purification of the fusion proteins. Forexample, the chimeric coding sequence can be modified to encode two ormore neighboring histidine residues, for example, in the amino orcarboxy terminus of the peptide. Histidine residue insertion can bereadily accomplished by the splice-by-overlap extension methodology, byincorporating histidine-encoding CAT and CAC triplet codons into the PCRprimers at suitable locations in the coding sequence. Histidine-modifiedproteins can be efficiently and quantitatively isolated bynickel-sepharose chromatography methods known in the art.

In some embodiments, therapeutics provided herein may also be conjugatedto a second molecule, such as a therapeutic agent (e.g., a cytotoxicagent). For example, the therapeutic agent includes, but is not limitedto, an anti-tumor drug, a toxin, a radioactive agent, a cytokine, asecond antibody or an enzyme. Examples of cytotoxic agents include, butare not limited to ricin, ricin A-chain, doxorubicin, daunorubicin,taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, maytansinoids, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as ²¹²Bi,¹³¹I, ⁹⁰Y, and ¹⁸⁶Re. Suitable detectable markers include, but are notlimited to, a radioisotope, a fluorescent compound, a bioluminescentcompound, chemiluminescent compound, a metal chelator or an enzyme. Thetherapeutics of the invention may also be conjugated to an anti-cancerpro-drug activating enzyme capable of converting the pro-drug to itsactive form. See, for example, U.S. Pat. No. 4,975,287.

In some preferred embodiments, the secondary therapeutic agent has acomplementary mode of action with the primary therapeutic agent. Forexample, in some embodiments, the primary and secondary therapeuticagents act against different aspects of a signal transduction systeminvolved in the etiology of a cancer or other disease, leading toenhanced efficacy, fewer side effects, an improved therapeutic index,and/or other benefits relative to tailored therapeutics bearing theprimary and/or secondary therapeutic agents only. In some embodiments,the first therapeutic agent potentiates the second therapeutic agent, orvice versa, or the first and second therapeutic agents exhibit asynergistic enhancement in one or more aspects of treatment. Methods forassessing synergism, potentiation, and other combined pharmacologicaleffects are known in the art, and described, e.g., in Chou and Talalay,Adv Enzyme Regul., 22:27-55 (1984), incorporated herein by reference.

In some embodiments, the therapeutic agent is conjugated to an antibodyimmune effector component of the therapeutic. Techniques for conjugatingor joining therapeutic agents to antibodies are well known (see, e.g.,Arnon et al., “Monoclonal Antibodies For Immuno-targeting Of Drugs InCancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldet al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982)).

The therapeutics of the invention can be administered by any methodknown in the art that is suitable for protein pharmaceuticals, such asintravenous injection, intramuscular injection, topical administration,oral ingestion, rectal administration, and inhalation. Alternatively,the therapeutics of the present invention can be delivered directly tothe site of the malignancy. The therapeutic may be administered inadmixture with a pharmaceutically acceptable carrier. Any such carriercan be used according to the present methods, as long as compatibilityproblems do not arise. An effective amount of the present recombinantfusion protein should be administered to the patient. The term“effective amount” refers to that amount of the fusion protein needed tobring about the desired response.

In some preferred embodiments, therapeutics made according to methodsproviding herein inhibit proliferation and/or induces apoptosis of cellsbearing the epitope against which the epitope binding protein wasscreened. For example, in some embodiments, the epitope-binding portionof the therapeutic complex can bind with the epitopes expressed on thesurface of malignant cells, or other targets. Once bound, theIR-eliciting antibody component of the complex stimulates the immunesystem to attack and eliminate the tagged cells, while sparing thenormal cells, as illustrated in FIG. 8.

In various embodiments, the tailoring of individualized therapeuticsprovided herein for binding to patient- and/or disease-specific targetsis made possible by utilizing novel methods for linking proteins totheir corresponding mRNAs (as “cognate pairs”). In some preferredembodiments, protein libraries are prepared comprising a large number ofcognate pairs, and the libraries are screened for cognate pairs thatbind to a target of interest, such as the individualized targetsdescribed herein.

Various aspects of the present invention use modified tRNA and/or mRNAmolecules to link translated protein products to their correspondingmRNAs via a tRNA linker, forming a “cognate pair.” In severalembodiments, mRNAs having unknown sequences are expressed in an in vitrotranslation system, for example from an mRNA library, and theircorresponding proteins are screened for one or more desiredcharacteristics, such as binding to the target epitope-binding domain,or another ligand of interest, and/or selectivity over one or moreadditional ligands, such as epitopes displayed by healthy cells. Infurther embodiments, proteins and their linked nucleic acids identifiedin one or more rounds of selection are modified, for example throughnucleic acid evolution (FIG. 4), to produce proteins with enhancedaffinities for the target ligand. Proteins having desiredcharacteristics, such as a high affinity for the target ligand, can thenbe produced in large quantities using standard cloning techniques byisolating their corresponding mRNA from the protein-mRNA cognate pairs.

In some preferred embodiments, cognate pairs are formed using aeukaryotic in vitro translation system, such as rabbit reticulocytelysate (RLL), wheat germ, E. coli, or yeast lysate systems. However, itis understood by the skilled artisan that any in vitro translationsystem can be used, including in situ systems, as well as hybridsystems, which combine components of different systems. For example, insome embodiments, one or more prokaryotic factors are used in aeukaryotic translation system, such as translation suppressor proteins(see e.g., Geller and Rich Nature 283:41 (1980); Edwards et al PNAS88:1153 (1991); Hou and Schimmel Biochem 28:6800 (1989), all hereinincorporated by reference). In some embodiments, one or more tRNAs ortRNA analogs are charged in a prokaryotic system and then purifiedaccording to established methods (Lucas-Lenard and Haenni, PNAS 63:93(1969), herein incorporated by reference) for use in a eukaryoticsystem.

In various embodiments, proteins comprising cognate pairs are linked totRNA or a tRNA analog by the action of ribosomal peptidyl transferase.In some embodiments, proteins are linked to a stable aminoacyl tRNAanalog (SATA). In some embodiments, the SATA is a tRNA with an aminoacid or amino acid analog attached to its 3′ end via a stable bond,relative to the corresponding high-energy ester bond in the nativestructure. When the SATA recognizes a particular codon, for example viahydrogen bonding, and accepts a nascent peptide chain by the action ofthe ribosomal peptidyl transferase, the stable aminoacyl bond preventsthe detachment of the tRNA from the polypeptide by peptidyl transferase,and also preserves the tRNA-polypeptide structure during subsequentsteps.

In some embodiments, a SATA is created according to methods generallydescribed in Fraser and Rich, PNAS, 70:2671 (1973), herein incorporatedby reference, which involve the conversion of a tRNA, or tRNA analog, toa 3′-amino-3′-deoxy tRNA. This is accomplished by adding a3′-amino-3′-deoxy adenosine to the end of a native tRNA with tRNAnucleotidyl transferase after removing the native adenosine, and thencharging the modified tRNA with an amino acid with the respectiveaminoacyl tRNA synthetase (aaRS). In some embodiments, the aaRS chargesthe tRNA on the 3′, rather than the 2′, hydroxyl, linking the amino acidto the tRNA by a stable amide bond, rather than the usual labilehigh-energy ester bond. Thus, when the SATA accepts a peptide fromribosomal peptidyl transferase it will stably hold the peptide and beunable to donate it to another acceptor.

In certain embodiments, tRNAs aminoacylated via a 3′ amide bond may notcombine with the elongation factor EF-TU, which assists in binding tothe A site (e.g., Sprinzl and Cramer, Prog. Nuc. Acid Res. 22:1 (1979),herein incorporated by reference). Such modified tRNAs do, however, bindto the A site. This binding of 3′ modified tRNAs can be increased bychanging the Mg⁺⁺ concentration (Chinali et al., Biochem. 13:3001(1974), herein incorporated by reference). The appropriateconcentrations of and/or molar ratios of SATA and Mg²⁺ can be determinedempirically. For example, if the concentration or A site avidity of aSATA is too high, the SATA may compete with native tRNAs for non-cognatecodons, stalling translation. Alternatively, if the concentration or Asite avidity of SATA is too low, the SATA might fail to effectivelycompete with release factors, preventing it from stably accepting thenascent peptide.

While the elongation factor is also believed to aid in proofreadingcodon-anticodon recognition, the absence of this source of proofreadingwould not be expected to interfere with methods provided herein. Withoutbeing bound to a particular mechanism, it is believed that the errorrate in the absence of elongation factor and associated GTP hydrolysisis approximately 1 in 100 for codons one nucleotide away (Voet and Voet,Biochemistry 2^(nd) ed. pp. 1000-1002 (1995), John Wiley and Sons,herein incorporated by reference). In some preferred embodiments, UAA isused as the linking codon. UAA has 7 non stop codons that differ from itby one amino acid, which comprises 7/61, or about 11.5% of the non stopcodons. Thus, the probability of miscoding a given codon can beestimated as (0.01)(0.115)=1.15×10⁻³ miscodes per codon, or about onemiscode every 870 codons, a frequency that would not substantiallyimpair performance of various methods described herein. In someadditional embodiments, UAG can be used as the linking codon withoutsubstantial impairment due to the absence of elongation factor-mediatedproofreading.

In some embodiment, the SATA is a tRNA, or tRNA analog, with one or moremodified bases in the acceptor stem, or another region of the molecule.Various methods for producing tRNAs with acceptor stem modification areknown in the art, and are described, for example, in Sprinzl and Cramer,Prog. Nuc. Acid Res., 22:1 (1979), herein incorporated by reference. Insome embodiments, a tRNA is modified with a puromycin moiety, such thatthe tRNA mimics aminoacyl-Tyr tRNA and is incorporated into the nascentpolypeptide, terminating translation. In some embodiments, acceptorstem-modified tRNAs are formed from “transcriptional tRNA”, wherein thesequence of the tRNA itself, rather than post-transcriptionalprocessing, leads to the atypical and modified bases. TranscriptionaltRNAs are capable of functioning as tRNAs (see e.g., Dabrowski et al.,EMBO J. 14:4872, 1995; and Harrington et al., Biochem. 32: 7617, 1993,both herein incorporated by reference). Transcriptional tRNA can beproduced by methods known in the art, such as transcription, or byconnecting commercially available RNA sequences (e.g., from DharmaconResearch Inc., Boulder, Colo.) together, piece-wise as in FIG. X, or bysome combination of established methods. For example, with reference toFig. X, the 5′ phosphate and 3′ puromycin are commercially availableattached to oligoribonucleotides, which can be connected together usingT4 DNA ligase (e.g., Moore and Sharp, Science 256: 992, (1992), hereinincorporated by reference) or alternatively, T4 RNA ligase (Romaniuk andUhlenbeck, Methods in Enzymology 100:52 (1983), herein incorporated byreference).

Additional methods for producing modified tRNAs are known in the art,and are described, e.g., in Chinali et al., Biochem. 13:3001 (1974) andKrayevsky and Kukhanova, Prog. Nuc. Acid Res 23:1 (1979), both hereinincorporated by reference.

In some embodiments, the tRNA is a nonsense suppressor tRNA comprising amodified or unmodified tRNA, or tRNA analog, that recognizes a stopcodon or a pseudo-stop codon, preferably by codon-anticodon hydrogenbonding, such that translation is terminated when the nascent protein isattached to the tRNA by peptidyl transferase. In some embodiments, thenonsense suppressor tRNA has 3′ modifications and/or sequences thatconform to the Yarus extended anticodon rules (Yarus, Science218:646-652, 1982, herein incorporated by reference). A “pseudo stopcodon,” as defined herein, refers to a codon which, while not naturallya nonsense codon, prevents a message from being further translated. Apseudo stop codon can comprise a codon recognized by a “stable aminoacyltRNA analog,” or SATA, as described herein, or a codon for which tRNAbearing a complementary anticodon is substantially depleted or absent,such that translation is terminated when the absent tRNA is required,i.e. at the pseudo stop codon. One skilled in the art will appreciatethat are numerous ways to create a pseudo stop codon, as defined herein.

In some preferred embodiments, the tRNA is a native tRNA, linked to thenascent polypeptide via a native peptide bond. In some embodiments, theSATA is a tRNA that is unmodified at the 3′ end, but which may have oneor more modifications to the anticodon loop and/or other regions of themolecule. In various embodiments, the use of native tRNAs and/or tRNAsthat are unmodified at the 3′ end results in improvements in variousselection methods described herein, giving rise to quicker, lesserror-prone, more efficient, more cost-effective, and/or higher yieldmethods. While not being bound by a particular theory, it is believedthat, under certain conditions, puromycin (and similar linkers) canresult in lower yields due to interference with the interaction betweenelongation factor(s) and tRNAs.

In one embodiment of the invention, the crosslinker is an agent thatchemically or mechanically links two molecules together. In oneembodiment, the crosslinker is an agent that can be activated to formone or more covalent bonds with tRNA and/or mRNA. In one embodiment, thecrosslinker is a sulfur-substituted nucleotide. In another embodiment,the crosslinker is a halogen-substituted nucleotide. Examples ofcrosslinkers include, but are not limited to, 2-thiocytosine,2-thiouridine, 4-thiouridine, 5-iodocytosine, 5-iodouridine,5-bromouridine and 2-chloroadenosine, aryl azides, and modifications oranalogues thereof. In one embodiment, the crosslinker is psoralen or apsoralen analog.

In some preferred embodiments, a psoralen is monoadducted to a tRNA, ora tRNA analog (e.g., a 3′ modified SATA), for example by connecting apsoralen linked oligonucleotide (FIG. 3), or by monoadduction (FIG. 4),to a native or modified tRNA or tRNA analog, preferably to the anticodonor another site distinct from the linkage to the polypeptide. Whenirradiated with UV light of a desired wavelength, a covalent psoralencrosslink is formed between the SATA and mRNA, as described in moredetail below. In some embodiments, the anticodon or other portion of atRNA is derivatized with a non-psoralen moiety capable of forming acrosslink to the mRNA, such as 2-thiocytosine, 2-thiouridine,4-thiouridine 5-iodocytosine, 5-iodouridine, 5-bromouridine,2-chloroadenosine, aryl azides, and modifications or analogues thereof.These and other cross-linkers are known in the art, and are availablecommercially, for example from Ambion, Inc. (Austin, Tex.), Dharmacon,Inc. (Lafayeffe, Colo.), and other well-known manufacturers ofscientific materials.

In various embodiments, a SATA-polypeptide complex or a tRNA-polypeptidecomplex is linked to the mRNA encoding the polypeptide via a linkermoiety, which can be located on the mRNA and/or on the tRNA. In somepreferred embodiments, the mRNA comprises a cross-linker, preferably ator near a stop codon at the 3′-end of the transcript, and the mRNA-tRNAlinkage is mediated entirely by the mRNA-based cross-linker. In furtherembodiments, the tRNA is unmodified at its 3′-end (e.g., a nonsensesuppressor tRNA). In some preferred embodiments, an mRNA comprises apseudo-stop codon located at the end of the translatable reading frame.A pseudo-stop codon can be effectively placed at the end of a readingframe where the pseudo-stop codon is located at the 3′ end of the mRNA,where the translation system is depleted of tRNAs corresponding tocodons 3′ to the pseudo-stop codon, and/or where 3′-modified tRNAscorresponding to the pseudo-stop codon are used, rendering thetranscript untranslatable and incapable of activating release factors.In some embodiments, the mRNA comprises a stop codon corresponding to atRNA stop anticodon. Advantageously, a stop codon/anticodon pair selectsfor full-length transcripts. One skilled in the art will understand thatan mRNA not having a stop codon may also be used and that any codon ornucleic acid triplet may be used.

In some methods, a SATA is attached to the translated message by apsoralen cross link between the codon and anticodon. Psoralen crosslinks are preferentially made between sequences that containcomplementary 5′ pyrimidine-purine 3′ sequences, especially UA or TAsequences (Cimino et al., Ann. Rev. Biochem. 54:1151 (1985), hereinincorporated by reference). The codon coding for the SATA, or thelinking codon, can be PYR-PUR-X or X-PYR-PUR, so that several codons maybe used for the linking codon. Conveniently, the stop or nonsense codonshave this configuration. Using a codon that codes for an amino acid mayrequire minor adjustments to the genetic code, which could complicatesome applications. Therefore, in a preferred embodiment, a stop codon isused as the linking codon and the SATA functions as a nonsensesuppressor in that it recognizes the linking codon. One skilled in theart, however, will appreciate that, with appropriate adjustments to thesystem, any codon can be used.

In some preferred embodiments, the SATA or peptidyl-tRNA is cross-linkedto the translated mRNA, for example between the codon and anticodon, bya psoralen cross-link, or by a cross-link formed from the groupconsisting of: 2-thio cytosine, 2-thio uridine, 4thio uridine5-iodocytosine, 5-iodouridine, 5-bromouridine, 2-chloroadenosine, andaryl azides. Psoralen cross links are, in some embodiments,preferentially made between sequences that contain complementary 5′pyrimidine-purine 3′ sequences, especially UA or TA sequences (Cimino etal., Ann. Rev. Biochem. 54:1151 (1985), herein incorporated byreference). In some embodiments, non-psoralen crosslinkers or arylazides are used and in certain embodiments, are particularlyadvantageous because they are less stringent in their requirements andtherefore increase the possible codon-anticodon pairs.

In various embodiments, translation terminates when the nascent proteinis attached to a SATA by the peptidyl transferase and/or when the end ofthe reading frame is reached. When a large number of ribosomes are inthis position, the SATA and the mRNA are cross-linked by application ofUV light. In a preferred method, cross-linking is accomplished byforming a psoralen crosslink upon irradiation with UV light, preferablyin the range of 320 nm to 400 nm. Psoralens comprise a furan side and apyrone side, and they readily intercalate between complementary basepairs in double stranded DNA, RNA, and DNA-RNA hybrids (Cimino et al.,Ann. Rev. Biochem. 54:1151 (1985), herein incorporated by reference). Insome preferred embodiments, psoralen cross-linking forms monoadducts,described more fully below, that are either pyrone sided or furan sidedmonoadducts. Upon further irradiation, the furan sided monoadducts canbe covalently crosslinked to complementary base pairs, whereas thepyrone sided monoadducts cannot be further crosslinked. The formation offuran-sided psoralen monoadducts (MAf) is achieved according toestablished methods. In additional embodiments, psoralen can also beattached at the end of the reading frame of the message.

Methods for large scale production of purified MAf on oligonucleotidesare described in the literature (e.g., Speilmann et al., PNAS 89:4514,1992, herein incorporated by reference), as are methods that requireless resources, but have some non-cross-linkable pyrone sided psoralenmonoadduct contamination (e.g., U.S. Pat. No. 4,599,303; Gamper et al.,J. Mol. Biol. 197:349 (1987); Gamper et al., Photochem. Photobiol. 40:29(1984), both herein incorporated by reference). In several embodimentsof the current invention, psoralen labeling is accomplished by usingeither method. In a preferred embodiment, furan sided monoadducts willbe created using visible light, preferably in the range of approximately400 nm-420 nm, according to the methods described in U.S. Pat. No.5,462,733 and Gasparro et al., Photochem. Photobiol. 57:1007 (1993),both herein incorporated by reference. In one aspect of this invention,a SATA with a furan sided monoadduct or monoadducted oligonucleotidesfor placement on the 3′ end of mRNAs, along with a nonadducted SATA areprovided as the basis of a kit.

In one embodiment, the formation and reversal of monoadducts andcrosslinks are performed according to the methods of Bachellerie et al.(Nuc Acids Res 9:2207 (1981)), herein incorporated by reference. In apreferred embodiment, efficient production of monoadducts, resulting inhigh yield of the end-product, is accomplished using the methods ofKobertz and Essigmann, J. A. Chem. Soc. 1997, 119, 5960-5961 and Kobertzand Essigmann, J. Org. Chem. 1997, 62, 2630-2632, both hereinincorporated by reference.

Other methods for connecting the in RNA to its protein can be used, aswell as methods of phage display.

In several embodiments, appropriate concentrations of SATA and Mg⁺⁺ areused in the in vitro translation system in the presence of the mRNAmolecules, causing translation to cease when ribosomes reach a stop, orpseudo-stop, codon which permits the SATA to accept the peptide chain,as described above. After a short time, a substantial proportion and/ornumber of the stop or pseudo-stop codons are occupied by SATAs withinribosomes, and in some embodiments, the system is then irradiated withUV light to generate cross-links between the tRNA-polypeptides and theircorresponding mRNAs. In several embodiments, the ribosomes are releasedor denatured after cross-linking of to mRNA, preferably by the depletionof Mg⁺⁺ through dialysis, dilution, or chelation. One skilled in the artwill understand that other methods, including but not limited to,denaturation by changing the ionic strength, the pH, or the solventsystem can also be used to release cognate pairs from associatedribosomes and/or other translation factors.

In various embodiments, cognate pairs are selected based on one or moredesired characteristics. In some embodiments, the selection of cognatepairs is based upon the binding of a target cell, protein, and/or otherbiomolecule, as determined by any of a variety of established methods,including, but not limited to, arrays, affinity columns,immunoprecipitation, and the like. In some preferred embodiments,selection criteria are measured using a high throughput screeningprocedure. The selection can be positive or negative in variousembodiments, according to the desired characteristics of the therapeuticagent. In several preferred embodiments, mRNAs whose sequences areunknown are expressed (e.g., in the form of an mRNA library from apatient, tissue, or other source of interest) and linked to theirencoded polypeptides using methods described herein, and the cognatepairs are screened to select for desired properties. For example, insome preferred embodiments, cognate pairs are assayed for binding to aligand of interest, such as an Fab idiotype displayed by a malignantcell, or other surface characteristic of a target cell. mRNA can beisolated for proteins exhibiting the desired binding characteristics,and large quantities of the protein can be produced using standardmolecular cloning techniques known in the art. As described in moredetail herein, the protein of interest can then be incorporated into amodular therapeutic agent, for example to target the agent to a patient-and/or disease-specific target.

In various embodiments, the selected cognate pairs can be those that dobind well to a ligand or those that do not. For instance, for a proteinto accelerate a thermodynamically favorable reaction, i.e., act as anenzyme for that reaction, it should bind both the substrate and atransition state analog. However, the transition state analog should bebound much more tightly than the substrate. This is described by theequation

$\frac{k_{enzyme}}{k_{\phi \; {enzyme}}} = \frac{K_{trans}}{K_{subst}}$

where the ratio of the rate of the reaction with the enzyme, k_(enzyme)to the rate without, k_(enzyme), is equal to the ratio of the binding ofthe transition state to the enzyme K_(trans) over the binding of thesubstrate to the enzyme K_(subst) (Voet and Voet, Biochemistry 2^(nd)ed. p. 380, (1995), John Wiley.

In some preferred embodiments, proteins which compete poorly for bindingto the substrate but compete well for binding to the transition stateanalog are selected. Operationally, this may be accomplished by takingthe proteins that are easily eluted from a matrix with substrate orsubstrate analog bound to it and are the most difficult to remove frommatrix with transition state analog bound to it. By sequentiallyrepeating this selection and reproducing the proteins throughreplication and translation of the nucleic acid of the cognate pairs, animproved enzyme should evolve. Affinity to one entity and lack ofaffinity to another in the same selection process is used in severalembodiments of the current invention. In some additional embodiments,cognate pairs can be selected according to one or more properties of themRNA portion.

There are many methods known in the art for identifying epitopesexpressed by normal and malignant cells. For example, in someembodiments, a peptide microarray can be used to isolate cell-specificmarker peptides from a combinatorial library, as described, e.g., byAina et al, “Therapeutic Cancer Targeting Peptides,” Biopolymers66:184-199 (2002). In some preferred embodiments, the protein-mRNAcomplex library is reacted an isolated population of malignant cells,and the degree of binding to the malignant cell epitope is measuredrelative to binding observed against a population of normal cells. Insome embodiments, proteins are selected having substantial affinity formalignant cells, with substantially lower or no affinity for normalcells. For example, in some embodiments, polypeptides are identifiedthat bind an epitope or other target of interest with an affinity ofless than about 10 μM, preferably less than about 1 μM, more preferablyless than about 0.1 μM, and even more preferably less than about 10 nM.In some preferred embodiments, a polypeptide has an affinity for theepitope or other target of less than about 1 nM. Screening methods canbe carried out with the potential ligands (e.g., proteins linked totheir cognate mRNA) and targets (e.g., cells targeted for treatment) ina variety of orientations. For example, in some embodiments, malignantcells are presented on a planar surface, such as a glass slide, and areexposed to a solution containing mRNA-protein cognate pairs. Boundproteins (cognate pairs) can be detected via a variety of methods knownin the art. For example, in some embodiments, the cognate pairs arederivatized, preferably on the mRNA and/or the tRNA linker, with adetectable probe, such as a biotin moiety, which can then be detectedwith a secondary reporter probe, for example using avidin coatedmagnetic beads. The resultantidiotype-(protein:mRNA)-biotin-avidin-magnetic bead complex can beidentified with a Ventana 320 automated immunohistochemistry system(Ventana Medical Systems, Tucson, Ariz.), or a similar system, asdescribed, for example, in Davis et al., Clinical Cancer Research 5:611-615, (1999).

The method can further comprise providing a plurality of distinctnucleic acid-polypeptide complexes, providing a ligand with a desiredbinding characteristic, contacting the complexes with the ligand,removing unbound complexes, and recovering complexes bound to theligand.

Several methods of the current invention involve the evolution ofnucleic acid molecules and/or proteins. In some embodiments, suchmethods comprise amplifying the nucleic acid component (as RNA, orcorresponding cDNA) of the recovered complexes and introducing variationto the sequence of the nucleic acids, for example by error-prone PCR, asdescribed, e.g., in Cadwell et al., PCR Methods Appl., 2: 28 (1992),incorporated herein by reference, in vitro recombination, described,e.g., in U.S. Pat. No. 5,605,793, mutagenesis, described, e.g., in U.S.Pat. No. 5,830,721, “DNA shuffling,” described, e.g., in Coco et al.,Nat Biotechnol, 19 (4):354-9 (2001), and/or other methods known in theart. In some preferred embodiments, at least one amino acid substitutionis introduced at each position in the protein. In further embodiments,the method further comprises translating polypeptides from the amplifiedand varied nucleic acids, linking them together using tRNA, andcontacting them with the ligand to select another new population ofbound complexes. Several embodiments of the present invention useselected protein-mRNA complexes in a process of in vitro evolution,especially the iterative process in which the selected mRNA isreproduced with variation, translated and again connected to cognateprotein for selection.

The Replication Threshold

A nominal minimum number of replications for efficient evolution may beestimated using the following formulae. If there is a sequence which isn sequences in length, with a selective improvement r mutations awaywith a mutation rate of p, the probability of generating the selectiveimprovement on replication may be determined as follows:

For r=1, probability of a mutation at the right point, p, times theprobability that it mutated to the right one of the three nucleotidesthat are different from the starting point, 1/3, times the probabilitythat the other n−1 sites remain unmutated, (1−p)^((n-r)), or

$P_{r} = {\left( \frac{p}{3} \right)^{1}\left( {1 - p} \right)^{({n - 1})}}$

where, P=the probability of attaining a given change r mutations away.More generally, for all r values:

$P_{r} = {\left( \frac{p}{3} \right)^{r}\left( {1 - p} \right)^{({n - r})}}$

It is instructive to compare the chances of finding an advantage onemutation away with the chances three mutations away. This is because,given the triplet genetic code, any given codon can only change intonine other codons in one mutation. Indeed, it turns out that no codoncan actually change into nine other amino acid codes in one mutation.The maximum number of amino acids that can be accessed in one mutationis seven amino acids and there are only eight codons of the sixty-fourthat can do this. Most codons have five or six out of nineteen otheramino acids within one mutation. To reach all nineteen amino acids thatare different from the starting one requires, in general, threemutations. These three mutations cannot be sequential since the twointervening ones will not, in general, be selectively advantageous.Therefore we need to use steps that are, at least, three mutations insize (r=3) to use all 20 amino acids.

For a mutation rate of 0.0067, which is that reported for “error-pronePCR”, using a message of 300 nucleotides, which gives a short protein of100 amino acids:

P ₃=1.51×10⁻⁹

Therefore, one would expect to need a threshold of:

$\frac{1}{1.51 \times 10^{- 9}} = {6.64 \times 10^{8}}$

replications at that mutation rate to reasonably expect to reach thenext amino acid that is advantageous. This is not the replication to usesince the binomial expansion shows that over 1/3 of trials (actuallyabout 1/e) would not contain the given sequence with selectiveadvantage.

A poisson approximation for large n and small p for a given μ can becalculated so that we can compute the general term when n is, say, ofthe order 10⁹ and p is of the order 10⁻⁹. The general term of theapproximation is:

$\frac{\mu^{r}}{{r!}^{\mu}}$

An amplification factor of greater than approximately 6/P ensures thatevolution will progress with the use of all amino acids. This is usefulwhen the production of novel proteins precludes the use of “shuffling”of preexisting proteins.

Limits on Purification

Given a reversible binding where B and C compete for A:

$\begin{matrix}{{{{AB}A} + {B\mspace{40mu} {AC}A} + C}{k_{B} = {{\frac{\lbrack A\rbrack \lbrack B\rbrack}{\lbrack{AB}\rbrack}\mspace{31mu} k_{C}} = \frac{\lbrack A\rbrack \lbrack C\rbrack}{\lbrack{AC}\rbrack}}}} & \; \\{\lbrack B\rbrack = {k_{B}\frac{\lbrack{AB}\rbrack}{\lbrack A\rbrack}}} & (1) \\{\lbrack C\rbrack = {k_{C}\frac{\lbrack{AC}\rbrack}{\lbrack A\rbrack}}} & (2)\end{matrix}$

The total concentrations can be expressed as follows:

[B] _(T) =[B]+[AB]  (3)

[C] _(T) =[C]+[AC]  (4)

Dividing (3) by (4):

$\frac{\lbrack B\rbrack_{T} = {\lbrack B\rbrack + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {\lbrack C\rbrack + \lbrack{AC}\rbrack}}$

And substituting (1) and (2) for [B] and [C]:

$\frac{\lbrack B\rbrack_{T} = {{k_{B}\left\lbrack \frac{AB}{A} \right\rbrack} + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {{k_{C}\left\lbrack \frac{AC}{A} \right\rbrack} + \lbrack{AC}\rbrack}}$

Rearranging the equation gives the following results:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack \left( \frac{k_{B} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}{\lbrack{AC}\rbrack \left( \frac{k_{C} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}$

Canceling the [A]'s in the numerator and denominator:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack \left( {k_{B} + \lbrack A\rbrack} \right)}{\lbrack{AC}\rbrack \left( {k_{c} + \lbrack A\rbrack} \right)}$

Finally, rearranging the equation provides the following equation:

$\begin{matrix}{{\frac{\lbrack{AB}\rbrack}{\lbrack{AC}\rbrack} = \frac{\lbrack B\rbrack_{T}\left( {k_{C} + \lbrack A\rbrack} \right)}{\lbrack C\rbrack_{T}\left( {k_{B} + \lbrack A\rbrack} \right)}}\frac{\left( {k_{C} + \lbrack A\rbrack} \right)}{\left( {k_{B} + \lbrack A\rbrack} \right)}} & \left( {{Enrichment}\mspace{14mu} {Factor}} \right)\end{matrix}$

The above factor is termed the “Enrichment Factor”. The ratio of thetotal components is multiplied by this factor to calculate the ratio ofthe bound components, or the enrichment of B over C. The maximumenrichment factor is k_(C)/k_(B), when the [A] is significantly smallerthan k_(C) or k_(B). When [A] is significantly greater than k_(C) ork_(B), the enrichment is 1, that is, there is no enrichment of one overthe other.

The enrichment is limited by the ratio of binding constants. To enrich ascarce protein that is bound 100 times as strongly as its competitors,the ratio of that protein to its competitors is increased by 1 millionwith 3 enrichments. To enrich a protein that only binds twice asstrongly as its competitors, 10 enrichment cycles would gain only anenrichment of ˜1000.

By an exactly analogous method an enrichment factor of selectingproteins that bind least well can be shown:

-   -   In the equation:

$\frac{\lbrack C\rbrack}{\lbrack B\rbrack} = \frac{{k_{C}\lbrack C\rbrack}_{T}\left( {\lbrack A\rbrack + k_{B}} \right)}{{k_{B}\lbrack B\rbrack}_{T}\left( {\lbrack A\rbrack + k_{C}} \right)}$

-   -   The enrichment here is maximal at [A]>k_(A) or k_(B).

$\frac{k_{C}\left( {\lbrack A\rbrack + k_{B}} \right)}{k_{B}\left( {\lbrack A\rbrack + k_{C}} \right)}$

The following Examples illustrate various embodiments of the presentinvention and are not intended in any way to limit the invention.

Example 1 Production of the SATA

One skilled in the art will understand that the SATA can be produced ina number of different ways. The protocols described below in thefollowing examples can be used for SATAs that have both a puromycin anda crosslinker on the tRNA, or that have a puromycin on the tRNA and acrosslinker on the mRNA. Where the crosslinker is on the mRNA, Example4, below, provides guidance. The following protocol is also instructivefor Linking tRNA Analogs, in the sense that Linking tRNA Analogs also,in a preferred embodiments, have a crosslinker on the tRNA.

For example, in a preferred embodiment, three fragments (FIG. 1) werepurchased from a commercial source (e.g., Dharmacon Research Inc.,Boulder, Colo.). Modified bases and a fragment 3 with a pre-attachedpuromycin on its 3′ end and a PO4 on its 3′ end were included, all ofwhich were available commercially. Three fragments were used tofacilitate manipulation of the fragment 2 in forming the monoadduct.

Yeast tRNAA1a or yeast tRNAPhe were used; however, sequences can bechosen from widely known tRNAs or by selecting sequences that will forminto a tRNA-like structure. Preferably, sequences with only a limitednumber of U's in the portion that corresponds to the fragment 2 areused. Using a sequence with only a few U's is not necessary becausepsoralen preferentially binds 5′UA3′ sequences (Thompson J. F., et alBiochemistry 21:1363, herein incorporated by reference). However, therewould be less doubly adducted product to purify out if such a sequencewas used.

Fragment 2 was preferably used in a helical conformation to induce thepsoralen to intercalate. Accordingly, a complementary strand wasrequired. RNA or DNA was used, and a sequence, such as poly C to one orboth ends, was added to facilitate separation and removal aftermonoadduct formation was accomplished.

Fragment 2 and the cRNA were combined in buffered 50 mM NaCl solution.The Tm was measured by hyperchromicity changes. The two molecules werere-annealed and incubated for 1 hour with the selected psoralen at atemperature ˜10° C. less than the Tm. The psoralen was selected basedupon the sequence used. A relatively insoluble psoralen, such as 8 MOP,could be selected which has a higher sequence stringency but may need tobe replenished. A more soluble psoralen, such as AMT, has lessstringency but will fill most sites. Preferably, HMT is used. If afragment 2 is chosen that contains more non-target U's, a greaterstringency is desired. Decreasing the temperature or increasing ionicstrength by adding Mg++ was also used to increase the stringency. In apreferred embodiment, MG++was omitted and ˜400 mM NaCl solution wasused.

Following incubation, psoralen was irradiated at a wavelength greaterthan approximately 400 nm. The irradiation depends on the wavelengthchosen and the psoralen used. For instance, approximately 419 nm 20-150J/cm2 was preferably used for HMT. This process results in an almostentirely furan sided monoadduct.

Purification of a Monoadduct

The monoadduct was then purified by HPLC as described in Sastry et al,J. Photochem. Photobiol. B Biol. 14:65-79, herein incorporated byreference. The fact that fragment 2 was separate from fragment 3facilitated the purification step because, generally, purification ofmonoadducts ≧25 mer is difficult (Spielmann et al. PNAS 89: 4514-4518,herein incorporated by reference).

Ligation of Fragment 2 and 3

The fragment 2 was ligated to the fragment 3 using T4 RNA ligase. Thepuromycin on the 3′ end acted as a protecting group. This is done as perRomaniuk and Uhlenbeck, Methods in Enzymology 100:52-59 (1983), hereinincorporated by reference. Joining of fragment 2+3 to the 3′ end offragment 1 was done according to the methods described in Uhlenbeck,Biochemistry 24:2705-2712 (1985), herein incorporated by reference.Fragment 2+3 was 5′ phosphorylated by polynucleotide kinase and the twohalf molecules were annealed.

In an alternative method, significant quantities of furan sidedmonoadducted U were formed by hybridizing poly UA to itself andirradiating as above. The poly UA was then enzymatically digested toyield furan sided U which was protected and incorporated into a tRNAanalog by nucleoside phosphoramidite methods. Other methods of formingthe psoralen monoadducts include the methods described in Gamper et al.,J. Mol. Biol. 197: 349 (1987); Gamper et al., Photochem. Photobiol.40:29, 1984; Sastry et al, J. Photochem. Photobiol. B Biol. 14:65-79;Spielmann et al. PNAS 89:4514-4518, U.S. Pat. No. 4,599,303, all hereinincorporated by reference.

SATAs generated by the methods described above read UAG (anticodon CUA).Additionally, UAA or UGA was also used. In various embodiments, anymessage that had the stop codon that was selected as the “linking codon”was used.

Example 2 Production of Psoralenated Furan Sided Monoadducts

UV Light Exposure of RNA:DNA Hybrids

Equal volumes of 3 ng/ml RNA:cRNA hybrid segments and of 10 μg/ml HMTboth comprised of 50 mM NaCl were transferred into a new 1.5 ml cappedpolypropylene microcentrifuge tube and incubated at 37° C. for 30minutes in the dark. This was then transferred onto a new clean culturedish. This was positioned in a photochemical reactor (419 nm peakSouthern New England Ultraviolet Co.) at a distance of about 12.5 cm sothat irradiance was ˜6.5 mW/cm2 and irradiated for 60-120 minutes.

Removal of Low Molecular Weight Protoproducts

100 μl of chloroform-isoamyl alcohol (24:1) was pipetted and mixed byvortex. The mixture was centrifuged for 5 minutes at 15000×g in amicrocentrifuge tube. The chloroform-isoamyl alcohol layer was removedwith a micropipette. The chloroform-isoamyl alcohol extraction wasrepeated once again. Clean RNA was precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) ice cold absolute ethanol was added to themixture. The tube was centrifuged for 15 minutes at 15,000×g in amicrocentrifuge. The supernatant was decanted and discarded and theprecipitated RNA was redissolved in 100 μl DEPC treated water thenre-exposed to the RNA+8-MOP.

Isolation of the Psoralenated RNA Fragments Using HPLC

All components, glassware and reagents were prepared so that they wereRNAase free. The HPLC was set up with a Dionex DNA PA-100 packagecolumn. The psoralenated RNA:DNA hybrid was warmed to 4° C. Thepsoralenated RNA was applied to HPLC followed by oligonucleotideanalysis, as described in the following section entitled“Oligonucleotide Analysis by HPLC.” The collected fractions represented:

(SEQ ID NO: 1) 5′CUAGAΨCUGGAGG3′, where Ψ is pseudouridine Furan sided(SEQ ID NO: 2) 5′CUPsoralenAGAΨCUGGAGG3′ monoadducts (SEQ ID NO: 3)5′XXXXXCCUCCAGAUCUAGXXXXX3′ (SEQ ID NO: 4)5′XXXXXCCUCCAGAUCUPsoralenAGXXXXX3′

The fractions were stored at 4° C. in new, RNAase free snappedmicrocentrifuge tubes and stored at −20° C. if more than four weeks ofstorage were required.

Identification of the RNA Fragments Represented by Each Peak FractionCollected by HPLC Using Polyacrylamide Gel Electrophoresis (PAGE)

The electrophoresis unit was set up in a 4° C. refrigerator. A gel wasselected with a 2 mm spacer. Each 5 μl of HPLC fraction was diluted to10 μl with Loading Buffer. 10 μl of each diluted fraction was loadedinto appropriately labeled sample wells. The tracking dye was loaded ina separate lane and electrophoresis was run as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” After the electrophoresis run was complete,the electrophoresis was stopped when the tracking dye reached the edgeof the gel. The apparatus was disassembled. The gel-glass panel unit wasplaced on the UV light box. UV lights were turned on. The RNA bands wereidentified. The bands appeared as denser shadows under UV lightingconditions.

Extraction of the RNA from the Gel

Each band was excised with a new sterile and RNAase free scalpel bladeand transferred into a new 1.5 ml snap capped microcentrifuge tube. Eachgel was crushed against the walls of the microcentrifuge tubes with theside of the scalpel blade. A new blade was used for each sample. 1.0 mlof 0.3M sodium acetate was added to each tube and eluted for at least 24hours at 4° C. The eluate was transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tube with a micropipette. A new RNAasefree pipette tip was used for each tube and the RNA with ethanol wasprecipitated out.

Ethanol Precipitation

Two volumes of ice cold ethanol was added to each eluate thencentrifuged at 15,000×g for 15 minutes in a microcentrifuge. Thesupernatants were discharged and the precipitated RNA was re-dissolvedin 100 μl of DEPC treated DI water. The RNA was stored in themicrocentrifuge tubes at 4° C. until needed. The tubes were stored at−20° C. if storage was for more than two weeks. The following was orderof rate of migration for each fragment in order from fastest to slowest:

(SEQ ID NO:1) 5′CUAGAΨCUGGAGG3′ Furan sided (SEQ ID NO:2)5′CUPsoralenAGAΨCUGGAGG3′ monoadducts (SEQ ID NO: 3)5′XXXXXCCUCCAGAUCUAGXXXXX3′ (SEQ ID NO: 4)5′XXXXXCCUCCAGAUCUPsoralenAGXXXXX3′

The tubes containing the remainder of each fraction were labeled andstored at −20° C.

Ethanol Precipitation

RNA oligonucleotide fragments were precipitated, and all glassware wascleaned to remove any traces of RNase as described in the followingsection entitled “Inactivation of RNases on Equipment, Supplies, and inSolutions.” All solutions were stored in RNAase free glassware andintroduction of nucleases was prevented. Absolute ethanol was stored at0° C. until used. Micropipettes were used to add two volumes of ice coldethanol to nucleic acids that were to be precipitated in microcentrifugetubes. Capped microcentrifuge tubes were placed into the microfuge andspun at 15,000×g for 15 minutes. The supernatant was discarded andprecipitated RNA was re-dissolved in DEPC treated DI-water. RNA wasstored at 4° C. in microcentrifuge tubes until ready to use.

Ligation of RNA Fragments 2 and 3

All glassware was cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following was added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube using a 100-1000 μlpipette and a new sterile pipette tip was used for each solution:

Fragment 2 (3.0 nM) 125.0 μl Fragment 3 (3.0 nM) 125.0 μl Reactionbuffer 250.0 μl RNA T4 ligase (9-12 U/ml)   42 μl

Reaction Buffer

RNase free DI-water 90.00 ml Tris-HCl (50 mM) 0.79 g MgCl2 (10 mM) 0.20g DTT (5 mM) 0.078 g ATP (1 mM) 0.55 g pH to 7.8 with HCL RNase freeDI-water QS to 100.00 ml

The mixture was gently mixed and the RNA was melted by incubating themixture at 16° C. for one hour in a temperature controlled refrigeratedchamber. RNA was precipitated out of the solution immediately after theincubation was completed.

Alcohol Precipitation

Two volumes (˜1000 μl) of ice cold absolute ethanol were added to thereaction mixture. The microcentrifuge tube was placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernatant was decantedand discarded and the precipitated RNA was re-dissolved in 100 μl DEPCtreated water. The mixture was electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following was the order of rate ofmigration for each fragment in order from fastest to slowest:

a) Frag. 2 (SEQ ID NO: 5) 5′CUAGAΨCUGGAGG3′-OHPsoralen b) Frag. 3 (SEQID NO: 6) 5′UCCUGUGTΨCGAUCCACAGAAUUCGCACC-Puromycin c) Frag 2 + 3 (SEQID NO: 7) 5′CUPsoralenAGAYCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCA CCPuromycin

Each fraction was isolated by UV shadowing, the bands were cut out, theRNAs were eluted from the gels and the RNA elute was precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure was repeated with any residual unligated fragment 2 and 3fractions. The ligated fractions 2 and 3 were pooled and stored in asmall volume of RNase free DI-water at 4° C.

Ligation of RNA Fragment 1 with Fragment 2+3

All glassware was cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following was added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube. A 100-1000 μL pipetteand new tip was used for each solution:

Fragment 2 + 3 (3.0 nM) 125.0 μl Reaction buffer 250.0 μl T4Polynucleotide Kinase(5-10 U/ml)  1.7 μl

Reaction Buffer

RNase free DI-water 90.00 ml Tris-HCl (40 mM) 0.63 g MgCl2 (10 mM) 0.20g DTT (5 mM) 0.08 g ATP (1 mM) 0.006 g pH to 7.8 with HCL RNase freeDI-water QS to 100.00 ml

The RNA was gently mixed then melted by heating the mixture to 70° C.for 5 minutes in a heating block. The mixture was cooled to roomtemperature over a two hour period and the RNA was allowed to anneal ina tRNA configuration. The RNA was precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) of ice cold absolute ethanol were added to thereaction mixture. The microcentrifuge tube was placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernatant was decantedand discarded and the precipitated RNA was re-dissolved in 100 μl DEPCtreated water. The mixture was electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following was the order of rate ofmigration for each fragment in order from fastest to slowest:

a) Frag. 1 (SEQ ID NO: 8) 5′GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACU3′ b) Frag2 + 3 (SEQ ID NO: 6) 5′CUPsoralenAGAYCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACC Puromycin c) Frag. 1 + 2 + 3 (SEQ ID NO: 9)5′GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUCUPsoralenAGAΨCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin

Each fraction was isolated by UV shadowing, the bands were cut out, theRNAs were eluted from the gels and the RNA elute was precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure was repeated with the unligated Fragment 1 and the 2+3Fraction. The ligated fractions 2+3 were pooled and stored in a smallvolume of RNase free DI-water at 4° C.

Final RNA Ligation

The following was added to a new 1.5 ml polypropylene snap cappedmicrocentrifuge tube. A 100-1000 μl pipette and new tip was used foreach solution:

Fragment 1 + 2 + 3 (3.0 nM) 250 μl reaction buffer 250 μl RNA T4 ligase(44 μg/ml)  22 μg

The mixture was incubated at 17° C. in a temperature controlledrefrigerator for 4.7 hours. Immediately after the incubation the tRNAwas precipitated out as described in step 6.2 above and the tRNA wasisolated by electrophoresis as described in the following sectionentitled “Polyacrylamide Gel Electrophoresis (PAGE) of Psoralenated RNAFragments.” The tRNA was pooled in a small volume of RNase free waterand stored at 4° C. for up to two weeks or stored at −20° C. for periodslonger than two weeks.

Polyacrylamide Gel Electrophoresis (Page) of Psoralenated RNA Fragments

Acrylamide Gel Preparation

All reagents and glassware were made RNAase free as described in thefollowing section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The gel apparatus was assembled to producea 4 mm thick by 20 cm×42 cm square gel. 29 parts acrylamide with 1 partammonium crosslinker were mixed at room temperature with the appropriateamount of acrylamide solution in an RNAase free, thick walled Erlenmeyerflask.

Acrylamide Solution

urea (7M) 420.42 g TBE (1X) QS to 1 L 5X TBE 0.455 M Tris-HCl 53.9 g 10mM EDTA 20 ml of 0.5M RNAase free DI water 900 ml pH with boric acid topH 9 QS with RNAase free DI water to 1 L

The mixture was degassed with vacuum pressure for one minute. Theappropriate amount of TEMED was added, mixed gently, and then the gelmixture was poured between the glass plates to within 0.5 cm of the top.The comb was immediately inserted between the glass sheets and into thegel mixture. An RNAase free gel comb was used. The comb produced wellsfor a 5 mm wide dye lane and 135 mm sample lanes. The gel was allowed topolymerize for about 30-40 minutes then the comb was carefully removed.The sample wells were rinsed out with a running buffer using amicropipette with a new pipette tip. The wells were then filled withrunning buffer.

Sample Preparation

An aliquot of the sample was suspended in loading buffer in a snapcapped microcentrifuge tube and vortex mixed. Indicator dye was notadded to the sample.

Loading Buffer

Urea (7M) 420.42 g Tris HCl (50 mM)  7.85 g QS with RNAase free D-H2O to1 L

Electrophoresis Run

The maximum volume of RNA/loading buffer solution was loaded into the135 mm sample wells and the appropriate volume of tracking dye in 5 mmtracking lane. The samples were electrophoresed in a 5° C. refrigerator.The electrophoresis was stopped when the tracking dye reached the edgeof the gel. The apparatus was then disassembled. Glass panels were notremoved from the gel. The gel-glass panel unit was placed on a UV lightbox. With UV filtering goggles in place, the UV lights were turned on.The RNA bands were identified. They appeared as denser shadows under UVlighting conditions. The RNA was extracted from the gel. Each band wasexcised with a new sterile and RNAase free scalpel blade and each bandwas transferred into a new 1.5 ml snap capped microcentrifuge tube. Eachgel was crushed against the walls of the microcentrifuge tubes with theside of the scalpel blade. A new blade was used for each sample. 1.0 mlof 0.3M sodium acetate was added to each tube and eluted for at least 24hours at 4° C. The eluate was transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tubes with a micropipette with a newRNAase free pipette tip for each tube. Two volumes of ice cold ethanolwas added to each eluate, then centrifuged at 15,000×g for 15 minutes ina microcentrifuge. The supernatants were discarded and the precipitatedRNA was redissolved in 100 μl of DEPC treated DI water. The RNA wasstored in the microcentrifuge tubes at 4° C. until needed.

Oligonucleotide Analysis by HPLC

HPLC purification of the RNA oligonucleotides is best effected usinganion exchange chromatography. Either the 2′-protected or 2′-deprotectedforms can be chromatographed. The 2′-protected form offers the advantageof minimizing secondary structure effects and provides resistance tonucleases. If the RNA is fully deprotected, sterile conditions arerequired during purification.

Deprotection of 2′-Orthoester Protected RNA

The tubes are centrifuged at 15,000×g for 30 seconds or until the RNApellet is at the bottom. 4001 of pH 3.8 deprotection buffer is added toeach tube of RNA.

Deprotection Buffer

Acetic acid (100 mM) is adjusted to pH 3.8 withtetramethylethylenediamine (TEMED). The pellet is completely dissolvedin the buffer by drawing in and out of a pipette. The tubes are vortexedfor 10 seconds and centrifuged at 15,000×g. The tubes are incubated in a60° C. water bath for 30 minutes. The samples are lyophilized beforeuse.

HPLC Column Conditions

A 4×250 mm column (DNAPAC PA, No. 043010) packed with Dionex(800)-DIONEX-0 (346-6390), with a capacity of 40 optical density units(ODU) at 260 nm is installed. The column temperature is set to 54° C.The injection volume is adjusted such that 5 μl produces approximately0.20 ODU.

Elution Buffers

Condition Buffer A Buffer B Sodium perchlorate (5 mM) 2.8 g (300 mM)168.0 g Tris-HCl 2.4 g 2.4 g Acetonitrile (2%) 80.0 ml 80.0 ml DI Water3900 ml 900 ml Adjusted pH 8.0 with HCL 8.0 with HCL q.s. 4000 ml 4000ml

HPLC Gradient

A 30% to 60% gradient of Buffer B for oligos 17-32 base pairs long isprovided:

Time Flow (minutes) (ml/min) % A % B Curve 0 1.5 100 0 * 1 1.5 100 0 6 31.5  70* 30* 6 15 1.5  40* 60* 6 15.5 2.5  0 100  6 17 2.5  0 100  617.25 2.5 100 0 6 23 2.5 100 0 6 23.1 1.5 100 0 6 24 1.5 100 0 6 25 0.1100 0 6 *% values that can be changed to modify the gradient. Typicalgradients are 0-30%, 20-50%, 30-60%, and 40-70% of Buffer B.

Gradient Selection

The gradient is selected based upon the number of bases, as follows:

Number of bases Gradient 0-5  0-30  6-10 10-40 11-16 20-50 17-32 30-6033-50 40-70 >50 50-80

After HPLC, the target samples are collected and the RNA concentrationis determined with a spectrophotometer at 260 nm. The samples are storedat −70° C.

Inactivation of RNAses on Equipment, Supplies, and in Solutions

Glassware was treated by baking at 180° C. for at least 8 hours.Plasticware was treated by rinsing with chloroform. Alternatively, allitems were soaked in 0.1% DEPC.

Treatment with 0.1% DEPC

0.1% DEPC was prepared. DI water was filtered through a 0.2 μM membranefilter. The water was autoclaved at 15 psi for 15 minutes on a liquidcycle. 1.0 g (wt/v) DEPC/liter of sterile filtered water was added.

Glass and Plasticware

All glass and plasticware was submerged in 0.1% DEPC for two hours at37° C. The glassware was rinsed at least 5× with sterile DI water. Theglassware was heated to 100° C. for 15 minutes or autoclaved for 15minutes at 15 psi on a liquid cycle.

Electrophoresis Tanks Used for Electrophoresis of RNA

Tanks were washed with detergent, rinsed with water then ethanol and airdried. The tank was filled with 3% (v/v) hydrogen peroxide (30 ml/L) andleft standing for 10 minutes at room temperature. The tank was rinsed atleast 5 times with DEPC treated water.

Solutions

All solutions were made using Rnase free glassware, plastic ware,autoclaved water, chemicals reserved for work with RNA and RNase freespatulas. Disposable gloves were used. When possible, the solutions weretreated with 0.1% DEPC for at least 12 hours at 37° C. and then heatedto 100° C. for 15 minutes or autoclaved for 15 minutes at 15 psi on aliquid cycle.

RNA Translation

2 μl of gastroinhibitory peptide (GIP) mRNA at a concentration of 20μl/ml was placed in a 250 μl snapcap polypropylene microcentrifuge tube.35 μl of rabbit reticulocyte lysate (available commercially fromPromega) was added. 1 μl of amino acid mixture which did not containmethionine (available commercially from Promega) was added. 1 μl of ³⁵Smethionine or unlabeled methionine was added. 2 μl of ³²P GIP mRNA orunlabeled GIP mRNA was added. Optionally, 2 ml of luciferase may beadded to some tubes to serve as a control. In a preferred embodiment,luciferase was used instead of GIP mRNA. One skilled in the art willunderstand that indeed any mRNA fragment containing the appropriatesequences may be used.

SATA was added to the experimental tubes. Control tubes which did notcontain SATA were also prepared. The quantity of SATA used wasapproximately between 0.1 μg to 500 μg, preferably between 0.5 μg to 50μg. 1 μl of Rnasin at 40 units/ml was added. Nuclease free water wasadded to make a total volume of 50 μl.

For proteins greater than approximately 150 amino acids, the amount oftRNA may need to be supplemented. For example, approximately 10-200 μgof tRNA may be added. In general, the quantity of the SATA should behigh enough to effectively suppress stop or pseudo stop codons. Thequantity of the native tRNA must be high enough to out compete the SATAwhich does not undergo dynamic proofreading under the action ofelongation factors.

Each tube was immediately capped, parafilmed and incubated for thetranslation reactions at 30° C. for 90 minutes. The contents of eachreaction tube was transferred into a 50 μl quartz capillary tube bycapillary action. The SATA was crosslinked with mRNA by illuminating thecontents of each tube with 2-10 J/cm2˜350 nm wavelength light, as perGasparro et al. (Photochem. Photobiol. 57:1007 (1993), hereinincorporated by reference). Following photocrosslinking, the contents ofeach tube were transferred into a new snapcap microfuge tube. Theribosomes were dissociated by chelating the calcium cations by adding 2μl of 10 mM EDTA to each tube. Between each step, each tube was gentlymixed by stirring each component with a pipette tip upon addition.

The optimal RNA for a translation was determined prior to performingdefinitive experiments. Serial dilutions may be required to find theoptimal concentration of mRNA between 5-20 μg/ml.

SDS-Page electrophoresis was performed on each sample, as describedabove. Autoradiography on the gel was performed, as described bySambrook et. al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.,Coldspring Harbor Press (1989), herein incorporated by reference.

The above example teaches the production and use of SATA (e.g.,puromycin on tRNA plus crosslinker on the tRNA) and the production anduse of Linking tRNA Analog (e.g., no puromycin, but has crosslinker ontRNA).

In another example, the SATA was produced in a manner similar to theabove methodology, except that uridines were substituted withpseudouridines. Substitution by pseudouridines can also be used withLinking tRNA Analog, as it facilities the formation of crosslinkermonoadduct formation (such as formation of the psoralen monoadduct).This technique is discussed below in Example 2.

Example 3 Production of the SATA Using Pseudouridine

As discussed above, one skilled in the art will appreciate that theSATA, Linking tRNA Analog and Nonsense Suppressor tRNA can be producedin a number of different ways. FIG. 5 shows the chemical structures foruridine and pseudouridine. Pseudouridine is a naturally occurring basefound in tRNA that forms hydrogen bonds just as uridine does, but lacksthe 5-6 double bond that is the target for psoralen. Pseudouridine, asused herein, shall include the naturally occurring base and anysynthetic analogs or modifications. In a preferred embodiment, the SATAwas produced using pseudouridine. Linking tRNA Analog can also beproduced using pseudouridine. Specifically, in a preferred embodiment,three fragments (FIG. 1) were purchased from a commercial source(Dharmacon Research Inc., Boulder, Colo.). Modified bases and a fragment3 (“Fragment 3”) with a pre-attached puromycin on its 3′ end and a PO₄on its 3′ end were included, all of which are available commercially.The three fragments were used to facilitate manipulation of a fragment 2(“Fragment 2”) in forming the monoadduct. Sequences of the threefragments, according to some embodiments, are as follows (2 examplesequences are provided for each fragment):

Fragment 1 (SEQ ID NO: 10) 5′PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACOH3′(SEQ ID NO: 16) 5′PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACOH3′ Fragment 2(SEQ ID NO: 11) 5′OHΨCUAACΨCOH3′ (SEQ ID NO: 17) 5′ OHΨCUAAAΨCOH 3′Fragment 3 (SEQ ID NO: 12) 5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin3′ (SEQ ID NO: 18) 5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin3′

The above sequences listed in Fragment 3 are applicable for SATA. ForLinking tRNA Analogs, the sequences would be similar, except thepuromycin would be replaced by adenosine.

Modified yeast tRNAA1a or yeast tRNAPhe was used according to oneembodiment of the invention. However, one skilled in the art willunderstand that sequences can be chosen widely from known tRNAs or byselecting sequences that will form into a tRNA-like structure. Oneadvantage of using pseudouridine in some embodiments is that thepseudouridine in Fragment 2 avoids psoralen labeling of the nontargetU's. Use of pseudouridine instead of uridine decreases the avidity ofthe A site of the ribosome for the tRNA analog but eliminates theinteraction of the terminal uridine with psoralen. The use of the Yarus“extended anticodon” guidelines increases A site binding (Yarus, Science218:646-652, 1982, herein incorporated by reference).

In one embodiment, Fragment 2 was used in a helical conformation toinduce the psoralen to intercalate. One skilled in the art willunderstand that other conformations can also be used in accordance withseveral embodiments of the invention. A complementary strand was alsoused. RNA or DNA was used, and a sequence, such as poly C or poly G whenC interacts with the psoralen to one or both ends, was added tofacilitate separation and removal after monoadduct formation wasaccomplished. Use of pseudouridine instead of uridines in the complementpermitted the use of a high efficiency wave length, such as about 365nm, without fear of crosslinking the product. Irradiation was preferablyin the range of about 300-450 nm, more preferably in the range of about320 to 400 nm, and most preferably about 365 nm. Further, use ofpseudouridine left the furan-sided monoadduct in place on Fragment 2because the Maf is the predominate first step in the crosslinkformation.

The following cRNA sequences with pseudouridine were used according to apreferred embodiment of the present invention. One skilled in the artwill understand that substitutions and modifications of these sequences,and of the other sequences listed herein, can also be used in accordancewith several embodiments of the current invention. For example, for SEQID NO: 19, listed below, the sequence can also be

5′XXXXXXGAΨΨΨAGAXXXXXXX3′: (SEQ ID NO: 30) CCCΨCCAGAGΨΨAGACCC (SEQ IDNO: 13) 5′CCCCCCGAΨΨΨAGACCCCCCC3′ (SEQ ID NO: 19)

Step 1: Furan Sided Monoadduction of Psoralen to Fragment 2

The formation of a furan sided psoralen monoadduct with the targeturidine of Fragment 2 was performed as follows:

A reaction buffer was prepared as follows:

Tris HCL   25 mM NaCl  100 mM EDTA 0.32 mM pH 7.0

4′hydroxy methyl-4,5′,8′-triethyl psoralen (HMT) was then added to afinal concentration of 0.32 mM and equimolar amounts of fragment 2 andcRNA were added to a final molar ratio of fragment2:cRNA:psoralen=1:1:1000. A total volume of 100 μl was irradiated at atime.

The mixture of complementary oligos, HMT, psoralen was processed asfollows:

1) Heated to 85° C. for 60 sec followed by cooling to 4° C. over 15 min,using PCR thermocycler.

2) Irradiated for 20 min at 4° C., in Eppendorf UVette plastic cuvette,covered top with parafilm, laid on the top of UV lamp (1 mW/cm²multi-wavelength UV lamp (λ>300 nm) (UV L21 model λ 365 nm).

Steps 1 and 2 above were repeated 4 times to re-intercalate andirradiate HMT. After the second irradiation additional 10 μl of 1.6 mMHMT was added in total 100 μl reaction volume. After 4 cycles ofirradiation, the free psoralens were extracted with chloroform and alloligos (labeled and unlabeled) were precipitated with ethanol overnight(see precipitation step). A small aliquot was saved for gelidentification.

Step 2: Purification of HMT Conjugated Fragment 2 (2 Ma) Oligo by HPLC

1) The reaction mixture was dried with speed vacuum for 10 minutes andthen was dissolved with 2 μl of 0.1 M TEAA, pH 7.0 buffer.

0.1 M TEAA, pH 7.0 Buffer

Acetic Acid   5.6 ml Triethylamine 13.86 ml H₂0 (RNAase free)   950 mlpH adjusted to 7.0 with acetic acid and water added to 1 L

2) The sample was loaded onto a Waters Xterra MS C18, 2.5 μm, 4.5×50 mmreverse-phase column pre-equilibrated with buffer A (5% wt/wtacetonitrile in 0.1M TEAA, pH 7.0) The sample was eluted with a gradientof 0-55% buffer B (15% wt/wt acetonitrile in 0.1M TEAA, pH 7.0) tobuffer A over a 35 minute time frame at a flow rate of 1 ml/minute. Thecolumn temperature was 60° C. and the detection wave length, set by anarrow band filter, was 340 nm. Furan sided psoralen monoadduct absorbsat 340 nm but the RNA, and any pyrone sided monoadduct does not. Thebuffer solutions were filtered and degassed before use.

The 2 MA eluted at around 25-28 minutes at a buffer B concentration of40%. Unpsoralenated fragment 2 eluted before 8 minutes based onsubsequent gel electrophoresis analysis on collected fractions.

The column was washed with 100% acetonitrile for 5 minutes and wasre-equilibrated with buffer A for 15 minutes. All fractions were driedwith speed vacuum overnight.

The fractions containing the 2 MA were identified by the level ofabsorbance at 260 nm (RNA) and 330 nm (furan sided psoralen monoadductedRNA). This was done by redissolving the dried fractions with 120 μl ofRnase-free distilled water and the absorbance was measured with aspectrophotometer at 260 nm and 330 nm. The fractions with highabsorbance at both wavelengths were pooled then dried with speed vacuum.A small aliquot from each was saved for gel analysis.

The cross-linked products were analyzed on a denaturing 20% TBE-urea geland visualized by gel silver staining.

Step 3: Purification of HMT Conjugated Fragment 2 Oligo from cRNA byHPLC

The dried samples were pooled and then were dissolved with 0.5×TEbuffer. A sample of about 0.4 absorbance unit was loaded onto a DionexDNAPac PA-100 (4×250 mm) column which was pre-equilibrated with buffer C(25 mM Tris-HCl, pH 8.0) and the column temperature was 85° C. (anionexchange HPLC).

The oligos were eluted at a flow rate of 1 ml/min. with a concavegradient from 4% to 55% buffer D for 15 minutes followed by a convexgradient from 55% to 80% with buffer D for the next 15 minutes. Theoligos were washed with 100% buffer D for 5 min and 100% buffer C foranother 5 min at a flow rate of 1.5 ml/min; Fractions were collectedthat absorbed 260 nm light. 2 MA had a retention time (RT) of 16.2minutes and was eluted by 57% buffer D, and free fragment 2 had RT lessthan 16.6 minutes, and was eluted by 55% buffer D and free cRNA had RTgreater than 19.2 minutes. The fractions were collected that absorbed at254 or 260 nm. The collected fractions were dried with speed vacuumovernight. All solutions were filtered and degassed before use.

The solution used comprised the following:

-   -   C: 25 mM Tris-HCl pH 8.0;    -   D: 250 mM NaClO4 in 25 mM Tris pH 8.0 buffer.    -   TE: 10 mM Tris-HCl pH 8.0 with 1 mM EDTA

Step 4: Desalting, Precipitation and Collection of the Purified 2 MAOligo

The dried fractions were redissolved with 100 μl Rnase free distilledwater. 500 μl cool 100% ethanol with 0.5M (NH4)2CO3 was added and themixture was vortexed briefly. The mixture was then frozen on dry ice for60 minutes or stored at −20° C. overnight.

The samples were then brought to 4° C. and centrifuged at maximum speedin a microcentrifuge for 15 minutes. The position of the pellet wasnoted and the supernatant was decanted or removed by pipette. Care wastaken not to disturb pellet. If the pellet still contained salt, thisstep was repeated. The pellet was then washed with 70% pre-cooledethanol twice. The wet pellet was dried with speed vacuum for 15 min.Urea PAGE gel identified the right fractions for the next step.

Step 5: Ligation of 2 MA Oligo to Fragment 3 Oligo

The following steps were performed:

A. The Following Reagents and Instruments were Used:

Nuclease-Free Water (Promega)

polyethylene glycol (PEG8000 Sigma) 40% (wt/wt in water)

RNasin® Ribonuclease Inhibitor (Promega)

phenol:chloroform

1.5 ml sterile microcentrifuge tubes

100% ethanol

70% ethanol

Dry ice or −20° C. freezer

Microcentrifuge at room temperature and +4° C.

PCR thermocycler or water bath

B. The Following Reaction Conditions were Used:

50 mM Tris-HCl (pH 7.8)

10 mM MgCl2,

10 mM DTT

1 mM ATP

18-20% PEG

C. The Following Reaction Mixture was Assembled in a SterileMicrocentrifuge Tube:

Fragment 3 (Donor) 1 μl (6 μg) (Purified, when necessary, before usingas a donor)

2 MA (Acceptor) 1 μl (1.5 μg)

After adding 8 μl Rnase free dH2O 8 μl, the reactions were incubated at85° C. for 1 minute to relax the oligo secondary structure, then slowlycooled to 4° C., using a PCR machine thermocycler. The preheated tubewas placed on ice to keep cool and centrifuged briefly, then thefollowing was added:

10X Ligase Buffer 4 μl 10 mM ATP 4 μl Rnase Out or Rnasin(40 u/μl)Promega 0.5 μl   PEG, 40% (Sigma) 20 μl  T4 RNA Ligase (10 u/μl) (NEB) 1μl

Nuclease-free water was added to final Volume of 40 μl. The mixture wasincubate at 16° C. overnight (16 hr). The mixture was centrifugedbriefly and then was placed on ice.

D. Precipitation of Oligonucleotides:

60 μl DEPC RNase free distilled water was added to the mixture and then150 μl phenol/chloroform was added. The mixture was vortexed vigorouslyfor 30 seconds. The precipitate was then centrifuged out at maximumspeed in a microcentrifuge for 5 minutes at room temperature. Theaqueous phase was transferred to a new microcentrifuge tube (>95 μl).

To this was added 3 μl 5 mg/ml glycogen, and 500 μl pre-cooled 100%ethanol with 0.5M (NH4)2CO3 and the mixture was vortexed briefly andthen was frozen on dry ice for 60 minutes. At this point, it may bestored overnight at −20° C. The dried fractions were redissolved with100 μl Rnase-free distilled water, 500 μl cool 100% ethanol with 0.5M(NH4)2CO3 was added and vortexed briefly. This was then frozen on dryice for 60 minutes or stored at −20C overnight. The samples were thenbrought to 4° C. and centrifuged at maximum speed in a microcentrifugefor 15 minutes and supernatant removed by pipette. Care was taken not todisturb pellet. If the pellet still contained salt, this step wasrepeated once. The pellet was then washed with 70% pre-cooled ethanolseveral times. This was then centrifuged at maximum speed in amicrocentrifuge for 5 minutes at 4C. The ethanol was carefully removedusing a pipette. Centrifugation was repeated again to collect remainingethanol which was carefully removed. The wet pellet was dried with speedvacuum for 10 min. A small aliquot was collected for the gel analysis.For long term storage, the RNA was stored in ethanol at −20C. Care wastaken not to store the RNA in DEPC water.

Step 6: Purification of the Ligated Fragment 3 Oligo Complex

The dried sample was redissolved with 0.5×TE buffer and was loaded ontoa DNAPac PA-100 column which was equilibrated with buffer C. The columntemperature was 85° C. and the detector operated at 254 nm to identifyfractions with RNA and at 340 nm to identify fractions with 2 MaF. Theoligos were eluted with a convex gradient from 30% to 70% with buffer Dfor the first 20 minutes at a flow rate of 0.8 ml/min and followed witha linear gradient from 70% to 98% D for another 20 min at the same flowrate. The elution was completed by washing with 100% D for 7 min and100% C for another 10 min at 1.0 ml/min flow rate. The fractions weredetected with 254 or 260 nm wavelength light. The ligated oligos (2MA-fragment 3) were eluted after 34 min, by more than 90% buffer B.Fractions with 254 nm absorbance (A_(254 nm)>0.01) were collected anddried with speed vacuum overnight.

Step 7: Purified 2 MA-Fragment 3 Desalting and Precipitation

The dried fractions were re-dissolved with 100 μl Rnase free distilledwater, 500 μl cool 100% ethanol with 0.5M (NH4)2CO3 was added and themixture was vortexed briefly. The mixture was then frozen on dry ice for60 minutes or stored at −20C overnight.

The samples were brought to 4° C. and centrifuged at maximum speed in amicrocentrifuge for 15 minutes. The position of the pellet was noted andthe supernatant decanted or removed by pipette. Care was taken not todisturb pellet. If still containing salt, this step was repeated. Thepellet was then washed with 70% pre-cooled ethanol twice. The wet pelletwas dried with speed vacuum for 15 min.

Urea PAGE was performed to identify the ligated 2 MA-fragment-3 for usein the next step of ligating fragment 1 to the 2 MA-fragment-3 oligowhich completes the SATA linker.

Step 8: Preparation of SATA (or Other tRNA Molecule)

A. RNA Oligo 5′phosphorylation

1. Reagent and Instrument:

Nuclease-Free Water (Cat.#P1193 Promega)

RNasin® Ribonuclease Inhibitor (Cat#N2511 Promega)

Phenol:chloroform

Sterile microcentrifuge tubes

100% ethanol

70% ethanol

Microcentrifuge at room temperature and 4° C.

PCR thermalcycler or water bath

2. Assemble The Following Reaction Mixture in a Sterile MicrocentrifugeTube:

Component Volume Acceptor RNA <200 ng T4 ligase 10× Reaction Buffer* 4μl RNasin ® Ribonuclease Inhibitor (40 u/μl) 20 unit T4 kinase (9-12u/μl) 2 μl 10 mM ATP 4 μl Nuclease-Free Water to final volume 40 μl

Incubate at 37° C. for 30 minutes in a PCR thermocycler or water bath.For non-radioactive phosphorylation, use up to 300 pmol of 5′ termini ina 30 to 40 μl reaction containing 1×T4 Polynucleotide Kinase ReactionBuffer, 1 mM ATP and 10 to 20 units of T4 Polynucleotide Kinase.Incubate at 37° C. for 30 minutes. 1×T4 DNA Ligase Reaction Buffercontains 1 mM ATP and can be substituted in non-radioactivephosphorylations. T4 Polynucleotide Kinase exhibits 100% activity inthis buffer). Fresh buffer is required for optimal activity (in olderbuffers, loss of DTT due to oxidation lowers activity.

B. Annealing Fragment 1 and 2 MA-Fragment 3 Oligo Complex:

1. Reagents and Instruments:

PCR thermocycler instrument or water bath

100 μg/ml nuclease-free albumin

100 mM MgCl2

2. Assemble the Following Reaction Mixture in a Sterile MicrocentrifugeTube:

Acceptor RNA oligo (1E) <200 ng Donor RNA oligo (3G-2G ligated oligo)<200 ng (5′ phosphorylated oligo from step A)

Appropriate ratios are as follows: Acceptor oligo:Donor oligo (Fragment1:2 MA-Fragment 3) molar ratio should be 1:1.1 to avoid fragment 1self-ligation. MgCl₂ was added to T4 ligase buffer (50 mM Tris-HCl, pH7.8, 10 mM MgCl₂, 10 mM DTT and 1 mM ATP) to final 20 mM concentration.Add Rnase free albumin to final 5 μg/ml. The final volume should be nomore than 100 μl. The solution was heated to 70° C. for 5 min, then wascooled from 70° C. to 26° C. over 2 hours and cooled from 26° C. to 0°C. over 40 minutes. Incubate at 16° C. for 16 to 17 hours using PCRinstrument.

C. Ligation of Annealed Oligos

Annealed oligos <15 μl  10 mM ATP  2 μl 40% PEG 18 μl T4 ligase 10XBuffer  2 μl RNasin ® Ribonuclease Inhibitor (40 u/μl) 0.5 μl  T4 ligase(9-12 u/μl)(NEB)  1 μl Nuclease-Free Water to final volume 40 μl

D. Precipitating tRNA Fragment

After ligation, 50 μl DEPC water and 150 μl phenol: chloroform wereadded and vortexed vigorously for 30 seconds. This was then centrifugedat maximum speed in a microcentrifuge for 5 minutes at room temperature.The aqueous phase was transferred to a new microcentrifuge tube (˜100μl). To this was added 2 μl 10 mg/ml mussel glycogen, 10 μl 3M sodiumacetate, pH 5.2. This was mixed well. Then 220 μl 95% ethanol was addedand vortexed briefly. The mixture was then frozen on dry ice for 30minutes. At this point the mixture may be stored over night at −20° C.or one may proceed. In one embodiment, the RNA should preferably not bestored in DEPC water, but in ethanol, at −20° C.

Then the samples were brought to 4° C. and centrifuged at maximum speedin a microcentrifuge for 15 minutes. The position of the pellet wasnoted and the supernatant decanted or removed by pipette. Care was takennot to disturb pellet. The pellet was then washed with 70% pre-cooledethanol twice. After removing the ethanol, the wet pellet was dried witha speed vacuum for 15 min. The dried pellet was stored at −20° C., untilthe next step.

RNA Translation

A luciferase mRNA which was modified to have the stop codoncorresponding to that recognized by the anticodon of the SATA (in thepresent case UAG) was used in a standard Promega in vitro translationkit in the recommended 1 μl of concentration 1 μg/μl. One skilled in theart will understand that indeed any mRNA fragment containing theappropriate sequences may be used.

SATA was added to the experimental tubes. Control tubes which did notcontain SATA were also prepared. The quantity of SATA used wasapproximately between 0.1 μg to 500 μg, preferably between 0.5 μg to 50μg. 1 μl of Rnasin at 40 units/ml was added. Nuclease free water wasadded to make a total volume of 50 μl.

For proteins greater than approximately 150 amino acids, the amount oftRNA may need to be supplemented. For example, approximately 10-200 μgof tRNA may be added. In general, the quantity of the SATA should behigh enough to effectively suppress stop or pseudo stop codons. Thequantity of the native tRNA must be high enough to out compete the SATAwhich does not undergo dynamic proofreading under the action ofelongation factors.

Each tube was immediately capped, parafilmed and incubated for thetranslation reactions at 30° C. for 90 minutes. The contents of eachreaction tube was transferred into a 50 μl quartz capillary tube bycapillary action. The SATA was crosslinked with mRNA by illuminating thecontents of each tube with 2-10 J/cm2˜350 nm wavelength light, as perGasparro et al. (Photochem. Photobiol. 57:1007 (1993), hereinincorporated by reference). Following photocrosslinking, the contents ofeach tube were transferred into a new snapcap microfuge tube. Theribosomes were dissociated by chelating the calcium cations by adding 2μl of 10 mM EDTA to each tube. Between each step, each tube was gentlymixed by stirring each component with a pipette tip upon addition.

The optimal RNA for a translation was determined prior to performingdefinitive experiments. Serial dilutions may be required to find theoptimal concentration of mRNA between 5-20 μg/ml.

SDS-Page electrophoresis was performed on each sample, as describedabove. Autoradiography on the gel was performed, as described bySambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.,Coldspring Harbor Press (1989), herein incorporated by reference.

The above example is instructive for the production and use of SATA(puromycin on tRNA and crosslinker on tRNA) and for the production anduse of Linking tRNA Analog (no puromycin, with crosslinker on tRNA).

Example 4 Production of a tRNA Analog Using Ribonucleotides Modified toForm Crosslinkers: Use of Psoralen and Non-Psoralen Cross-Linkers

As described above, pseudouridine can be used in some embodiments tominimize the formation of unwanted monoadducts and crosslinks. In oneembodiment, a crosslinker modified mononucleotide is formed and used.One advantage of the crosslinker modified mononucleotide is that itminimizes the formation of undesirable monoadducts and crosslinks.

As discussed above, one skilled in the art will appreciate that theSATA, Linking tRNA Analog, and Nonsense Suppressor Analog can beproduced in a number of different ways. In a preferred embodiment,psoralenated uridine 5′ mononucleotide, 2-thiocytosine, 2-thiouridine,4-thiouridine 5-iodocytosine, 5-iodouridine, 5-bromouridine or2-chloroadenosine can be produced or purchased and enzymatically ligatedto an oligonucleotide to be incorporated into a tRNA analog. Arylazides, and analogues of aryl azides, and any modifications thereto, canalso be used in several embodiments, as a linking moiety or agent. Thefollowing protocol can be employed for crosslinkers that are located onthe tRNA. One skilled in the art will understand that this protocol canalso be used for crosslinkers located on the mRNA. Thus, the followingexample is instructive on the production and use of SATA, Linking tRNAAnalog, and Nonsense Suppressor Analog.

Production of Modified Nucleotide

4-thioU, 5-iodo and 5-bromo U with and without puromycin can bepurchased already incorporated into a custom nucleotide up to 80basepairs in length (Dharmacon, Inc). Therefore, the SATA, and theLinking tRNA Analog with these crosslinkers already in place, andsimilar crosslinkers, can be purchased directly from Dharmacon, Inc.Nonsense Suppressor Analog can also be purchased from Dharmacon, Inc.

2-thiocytosine, 2-thiouridine, 4thiouridine 5-iodocytosine,5-iodouridine, 5-bromouridine or 2-chloroadenosine can all be purchasedfor crosslinking from Ambion, Inc. for the use in the Ambion MODIscriptkit for incorporation into RNA. Therefore, the SATA and the Linking tRNAAnalog along with these crosslinkers, and similar crosslinkers, can bepurchased directly from Ambion, Inc

The PO₄U_(psoralen) can be produced as follows:

(SEQ ID NO: 20) AUAUAUAUAUAUAUAUAUAUGGGGGG (seq A1) (available fromDharmacon, Inc.) (SEQ ID NO: 21) CCCCCCATATATATATATATATATAT (seq A2)(available from University of Southern California services).

The formation of a furan-sided psoralen monoadduct with the targeturidine is performed as follows:

A reaction buffer is prepared. The reaction buffer, with a pH of 7.0,contains 25 mM Tris HCL, 100 mM NaCl, and 0.32 mM EDTA.

4′hydroxy methyl-4,5′,8′-triethyl psoralen (HMT) is then added to afinal concentration of 0.32 mM and equimolar amounts of seq A1 and seqA2 are added to a final molar ratio of seq A1:seq A2:psoralen=1:1:1000.A total volume of 100 μl is irradiated at a time.

The mixture of complementary oligos, HMT, trimethylpsoralen is processedas follows: 1) Heat to 85° C. for 60 sec followed by cooling to 4° C.over 15 min, using PCR thermocycler; and 2) Irradiate for 20 to 60 minat 4° C., in Eppendorf UVette plastic cuvette, covered top withparafilm, in an RPR-200 Rayonet Chamber Reactor equipped with a coolingfan and 419 nm wave. This is either placed on an ice water bath or in a−20° C. freezer.

Steps 1 and 2 above are repeated 4 times to re-intercalate and irradiateHMT. After 4 cycles of irradiation, the free psoralens are extractedwith chloroform and all oligos (labeled and unlabeled) are precipitatedwith ethanol overnight (see precipitation step). A small aliquot issaved for gel identification.

Comparable sequences can be produced using the Ambion, Inc kit fornon-psoralen crosslinkers.

RNase H Digestion of RNAs in DNA/RNA duplexes

The following steps are performed: (1) Dry down oligos in speed vac; (2)Resuspend pellet in 10 μL 1×Hyb Mix; (3) Heat at 68° C. for 10 minutes;(4) Cool slowly to 30° C. Pulse spin down; (5) Add 10 μL 2×RNase HBuffer. Mix. (6) Incubate at 30° C. for 60 minutes; (7) Add 130 μL StopMix.

For the Phenol/Chloroform extract: (1) Add 1 vol. phenol/chloroform; (2)Vortex well; (3) Spin down 2 minutes in room temperature microfuge; (4)Remove top layer to new tube.

For the Chloroform extract: (1) Add 1 vol. chloroform; (2) Vortex well;(3) Spin down 2 minutes in room temperature microfuge; (4) Remove toplayer to new tube.

Then, (1) Add 375 μL 100% ethanol; (2) Freeze at −80° C.; (3) Spin down10 minutes in room temperature microfuge; (4) Wash pellet with 70%ethanol; (5) Resuspend in 10 μL loading dye; (6) Heat at 100° C. for 3minutes immediately before loading.

Purification of monoribonucleotides nucleotides from the longer cDNA aswell as longer RNA fragments, is accomplished using anion exchange HPLC.The psoralen-monoadducted mononucleotides (PO₄U_(psoralen)) are thenseparated by reverse phase HPLC from mononucleotides that were notmonoadducted (PO₄U and PO₄A).

Similar digestion techniques and nucleotide incorporation, describedbelow, can also be used for non-psoralen crosslinkers using the Ambion,Inc kit.

Incorporation of Light Sensitive Nucleotides into the tRNA ComponentOligoribonucleotides

The following protocol can be used for incorporating a pU_(crosslinker)into a CUA stop anticodon. However, one skilled in the art willunderstand that other nucleotides can also be used to produce other stopanticodons and pseudo stop anticodons in accordance with the methodsdescribed herein

Generally, methods adapted from the protocols for T4 RNA ligase areused, but with some modification because of the lack of protection ofthe 3′ OH of the modified nucleotides.

5′OH CUC OH 3′ oligoribonucleotides (seq B1) can be purchased fromDharmacon, Inc. and can be as acceptors in the ligation. The molar ratioof B1 to psoralenated mononucleotides is preferably kept at 10:1 to 50:1so that the modified U's will be greatly out-numbered, therebypreventing the formation of CUC(U_(crosslinker))_(N). This makes one ofthe preferred reactions:

In one embodiment, the product is purified by sequential anion exchangeand reversed phase HPLC to ensure that the psoralenated U and the longerpsoralenated 7 mer are separated. The 7 mer is then 3′ protected byligation with pAp yielding CUCU_(crosslinker)Ap (Fragment 2B).

This is again purified with anion exchange HPLCF or the next ligation.

First Ligation of Fragment 2B to 1B or 1B1

This 2B fragment can be used in a tRNA analog that has a stable acceptoror one that has a native esterified acceptor. In one embodiment, toassure that the native 3′ end can be aminoacylated by native AA-tRNAsynthetases, the acceptor stem is modified in that version of theanalog. In the SATA version, in one embodiment, the 3′ fragment ismaintained with a commercially prepared puromycin as the acceptor. Thus,in one embodiment, the following are used in two different 5′ ends:

(SEQ ID NO: 22) 5′ OHGCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA 3′ seq 1B (to beused with the tRNA analog with the stable puromycin acceptor) and (SEQID NO: 23) 5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGA 3′ seq 1B₁ (to be usedwith the native esterified acceptor).

The ligation is performed again with T4 RNA ligase and purified bylength. The equation for sequence 1B is as follows:

For sequence 1B₁:

Ligation of the Two Half-Molecules of the tRNA Analog

The above product is treated with T4 polynucleotide kinase in twoseparate steps to remove the 3′ phosphate and add a 5′ phosphate.

The newly prepared 5′ and 3′ half molecules ends are then ligatedgenerally following the previous protocols. The 3′ sequencescorresponding to the respective 5′ sequences are as follows:

Sequence 1B: (Ψ = pseudouridine) (SEQ ID NO: 24)5′ PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(cross-) _(linker)A 3′corresponded to the 3′ half: (SEQ ID NO: 31)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPur 3′, 3B and sequence 1B1,(SEQ ID NO: 25) 5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(cross-)_(linker)APO₄ corresponded to 3′ half (SEQ ID NO: 32)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUCUCCACCA3′.

The latter is recognizable by the aminoacyl tRNA synthetase for alaninein E. coli.

The example described above can be used to make and use the SATA,Linking tRNA, and the Nonsense Suppressor tRNA.

Example 5 Placement of Crosslinkers on the mRNA for SATA and NonsenseSuppressor tRNA

In several embodiments, the crosslinker (such as psoralen or anon-psoralen crosslinker) is not placed on the tRNA, but rather locatedon the mRNA. For example, in one embodiment, the SATA comprises apuromycin located on the tRNA, while the crosslinker is on the mRNA. Inyet another embodiment, the Nonsense Suppressor tRNA is used, and thiscomprises a tRNA with no puromycin, with the crosslinker being on themRNA. Placement of the crosslinker on the message (the mRNA) can beaccomplished as set forth below. The relevant sequence is as follows:

(SEQ ID NO: 26) GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUG AAAAUG AAA AUG U_(crosslinker)AG

For convenience only, and in one embodiment, a message with both Kozakand Shine Dalgarno sequences that has a large number of methioninecodons for ³⁵S labeling is used.

For 4-thiouridine, 5-bromouridine and 5-iodouridine, the message can bepurchased fully-made from Dharmacon, Inc. For aryl azides, the methodrecited in Demeshkina, N, et al., RNA 6:1727-1736, 2000, hereinincorporated by reference, can be used.

For 2-thiocytosine, 2-thiouridine, 5-iodocytosine, or 2-chloroadenosine,the modified bases can be purchased as the 5′ monophosphate nucleotidefrom Ambion, Inc. When psoralen is used as the crosslinker, the modified5′ monophosphate nucleotide is made as above.

The modified 5′ monophosphate nucleotides are first incorporated intohexamers to facilitate purification. The construction of uridinecontaining crosslinkers is shown but in several embodiments, the otherbases can be incorporated into both stop and pseudo stop codons usingsimilar techniques:

was accomplished using a similar protocol described above, except apreponderance of AUG was used because of the absence of a 3′ protectionof the pNcrosslinker. The product was purified by anion exchange HPLCfrom the excess of AUG. Then 5′ pAGbiotin 3′ was added with T4 RNAligase. The 3′ biotin was simply a convenient 3′ blocking groupavailable form Dharmacon. The resulting AUGU_(crosslinker)AG_(biotin)was again purified followed by 5′ phosphorylation and ligated to:

(SEQ ID NO: 27) GGGUUAACUUUAGAAGGAGGUCGCCACCAUGGUUAAAAUGAAAAUGA AAAUGAAA(sequence M1) to produce (SEQ ID NO: 28)GGGUUAACUUUAGAAGGAGGUCGCCACCAUGGNNAAAAUGAAAAUGAAAAUGAAAAUGU_(crosslinker)AG_(biotin).

The yield is high enough to obviate purification. Accordingly, using theprotocol described above, SATAs and Nonsense Suppressor tRNAs can bemade and used in accordance with several embodiments of the presentinvention.

Example 6 Using tRNA Systems that do not Require Puromycin

Several embodiments of the present invention provide a system and methodthat do not require puromycin, puromycin analogs, or other amidelinkers. In one embodiment, Linking tRNA Analogs and Nonsense SuppressortRNAs do not require puromycin and can be made and used according to thefollowing example.

For systems without puromycin, a translation system to aminoacylate thetRNA can be used. In other embodiments, aminoacylation can beaccomplished chemically. One skilled in the art will understand how tochemically aminoacylate tRNA. Where translation systems are used, anytype of translation system for aminoacylation can be employed, such asin vitro, in vivo and in situ. In one embodiment, am e-coli translationsystem is used. An E. coli translation system is used for systems with atRNA modified to be recognized by the aaRS^(Ala). In one embodiment,this is preferable for systems without the stable acceptor (e.g. thepuromycin)

3 mcg of each of the following mRNA's are translated in 40 microliterseach of Promega S30 E. coli translation mixture:

(SEQ ID NO: 28) a) GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUGAAA AUG AAA AUGUcrosslinkerAGbiotin and (SEQ ID NO: 29) b)GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUG AAA AUG AAA AUGUAG

3 mcg of amber suppressor tRNA manufactured as above are added to thefirst.

3 mcg of suppressor with crosslinker on the anticodon are added to thesecond. 35S-methionine is added to both and the mixtures are thenincubated at 37° C. for 30 minutes. The reactions are then rapidlycooled by placement in an ice bath, transferred to a flat Petri dish andfloated in an ice bath so that the mixture is 1.5 cm below a ˜350 nmlight source. They are exposed at ˜20 J/cm for 15 min.

After irradiation, the mixtures are phenol extracted and ethanolprecipitated. In this manner, systems such as the Linking tRNA Analogsand Nonsense Suppressor tRNAs are aminoacylated and used to connect themessage (mRNA) to its coded peptide in accordance with severalembodiments of the present invention.

Example 7 Alternative Sequences

In a preferred embodiment, Fragments 1, 2 and 3, described above inExample 1, have the following alternate sequences:

Fragment 1 (SEQ ID NO: 13):

5′ PO4 GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA N3-Methyl- U 3′

Fragment 2 (SEQ ID NO: 14):

5′ UCUAAGΨCΨGGAGG 3′Fragment 3-Unchanged from the sequence listed above (SEQ ID NO: 6):

5′ PO4 UCCUGUGTΨCGAUCCACAGAAUUCGCACC Puromycin 3′

Using the methods described above, the sequence of alternative Fragments1+2+3 was (SEQ ID NO: 15):

5′PO4GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA(N3-MethylU)UCUPsoralenAAGΨCΨGGAGGUCCUGUGTYCGAUCCACAGAAUU CGPuromycin 3′

For linking tRNA Analog and Nonsense Suppressor tRNA, the abovesequences are similar, except adenosine is used to replace puromycin.

While a number of preferred embodiments of the current invention andvariations thereof have been described in detail, other modificationsand methods of use will be readily apparent to those of skill in theart. For all of the embodiments described above, the steps of themethods need not be performed sequentially. Accordingly, it should beunderstood that various applications, modifications and substitutionsmay be made without departing from the spirit of the invention or thescope of the claims.

1. A method for making a targeted therapeutic for treating a disease orcondition, the method comprising: obtaining a biological sample from apatient having the disease or condition, or who is at risk fordeveloping the disease or condition, the sample comprising a populationof diseased cells; screening a library comprising proteins linked totheir cognate mRNAs to identify mRNA-protein pairs that bind to thediseased cells; isolating one or more proteins from the identifiedmRNA-protein pairs; and conjugating the isolated protein(s) to atherapeutic agent, the therapeutic agent having therapeutic efficacyagainst the diseased cells.
 2. The method of claim 1, furthercomprising: isolating a first sub-population of cells from the sample,the first sub-population comprising diseased cells substantially freefrom non-diseased cells; and isolating a second sub-population of cellsfrom the sample, the second population comprising non-diseased cellssubstantially free of diseased cells, wherein the library is screened toidentify mRNA-protein pairs that bind the diseased cells withsubstantially higher affinity than the non-diseased cells
 3. The methodof claim 1, wherein the disease or condition is a cancer, and thediseased cells are malignant cells.
 4. The method of claim 3, whereinthe disease or condition is a hematological cancer, and the malignantcells comprise lymphocytes.
 5. The method of claim 4, wherein themalignant cells are B-cells.
 6. The method of claim 4, wherein themalignant cells are T-cells.
 7. The method of claim 1, wherein thedisease or condition is a pathogenic infection, and the diseased cellsare cells infected with a pathogen.
 8. The method of claim 7, whereinthe pathogen is a virus.
 9. The method of claim 8, wherein the virus isHIV.
 10. The method of claim 1, wherein the therapeutic agent is acytotoxic agent.
 11. The method of claim 1, wherein the therapeuticagent is capable of effecting an immune response in the patient.
 12. Themethod of claim 11, wherein the therapeutic agent is an adjuvant. 13.The method of claim 1, wherein the disease or condition is transplantrejection, and the diseased cells are transplanted cells.
 14. The methodof claim 13, wherein the therapeutic agent is capable of suppressing animmune response in the patient.
 15. The method of claim 1, wherein thedisease or condition is transplant rejection, and the diseased cells arelymphocytes that bind to transplanted cells.
 16. The method of claim 15,wherein the therapeutic agent is capable of effecting an immune responsein the patient.
 17. The method of claim 14, wherein the therapeuticagent stimulates complement-mediated immune response.
 18. The method ofclaim 17, wherein the therapeutic agent is C3 convertase.
 19. The methodof claim 1, wherein the isolating step comprises cloning anoligonucleotide corresponding to the mRNA of the identified mRNA-proteinpair, and expressing protein from the oligonucleotide.
 20. (canceled)21. The method of claim 18, wherein the cross-linker is placed on acodon.
 22. The method of claim 18, wherein the cross-linker is placed ona pseudo-stop codon.
 23. The method of claim 18, wherein thecross-linker comprises a psoralen cross-linker, and the linkingcomprises exposing the mRNA to UV light.
 24. The method of claim 18,wherein the translating is performed in vitro.
 25. The method of claim18, wherein at least one of the candidate mRNA molecules and at leastone the translated proteins is linked by a tRNA molecule selected fromthe group consisting of tRNA, modified tRNA, and tRNA analogs.
 26. Themethod of claim 18, wherein the disease or condition is a cancer, andthe diseased cells are malignant cells.
 27. The method of claim 24,wherein the disease or condition is a hematological cancer, and themalignant cells comprise lymphocytes.
 28. The method of claim 18,wherein the disease or condition is a pathogenic infection, and thediseased cells are cells infected with a pathogen.
 29. The method ofclaim 18, wherein the disease or condition is transplant rejection, andthe diseased cells are transplanted cells.
 30. The method of claim 27,wherein the therapeutic agent is capable of suppressing an immuneresponse in the patient.
 31. The method of claim 18, wherein the diseaseor condition is transplant rejection, and the diseased cells arelymphocytes that bind to transplanted cells
 32. The method of claim 29,wherein the therapeutic agent is capable of effecting an immune responsein the patient.
 33. The method of claim 18, wherein the therapeuticagent is a cytotoxic agent.
 34. The method of claim 18, wherein thetherapeutic agent is capable of effecting an immune response in thepatient. 35-43. (canceled)